A method for specific transmitter identification of retrogradely labeled neurons: Immunofluorescence combined with fluorescence tracing

Brain Research Reviews, 8 (1984) 99-127 Elsevier

99

BRR 90018

A Method for Specific Transmitter Identification of Retrogradely Labeled Neurons: Immunofluorescence Combined with Fluorescence Tracing L. SKIRBOLL’, T. HGKFELT ‘J-3, G. NORELL’.3, 0. PHILLIPSON4,*, H. G. J. M. KUYPERS, M. BENTIVOGLIO”.**, C. E. CATSMAN-BERREVOETS, T. J. VISSER6, H. STEINBUSCH7, A. VERHOFSTADs. A. C. CUELLO’, M. GOLDSTEINi and M. BROWNSTEINt ‘Clinical Neuroscience Branch and Laboratory of Clinical Science, NIMH, ZFogarty International Center, NIH, Bethesda, MD (U.S.A.) and Department of3Histology and 4Anatomy, Karolinska Institute, Stockholm (Sweden); SDepartments of Anatomy and “Internal Medicine and Clinical Endocrinology, Erasmus University, Rotterdam, ‘Department of Pharmacology, Free University, Amsterdam, XDepartment of Anatomy and Embryology, Catholic University, Nijmegen (The Netherlands); YDepartment of Anatomy, University of Oxford, Oxford (G. B.) and JODepartment of Psychiatry, New York University, New York, NY (U.S.A.)

(Accepted September 4th, 1984) Key words: retrograde tracing -

immunohistochemistry

- fluorescent dyes - brain pathways-collateral

tracing - rats

CONTENTS 1. Introduction

.............................................................................................................................................

100

2. Experimental procedures ............................................................................................................................. 2.1. Dyes ................................................................................................................................................ 2.2. Injection procedures and transport time .................................................................................................... 2.3. Immunocytochemistry .......................................................................................................................... 2.4. Visualization and Evaluation .................................................................................................................. 2.5. Studies on Collaterals ...........................................................................................................................

101 101 102 102 102 103

3. Results .................................................................................................................................................... 3.1. Dye injection sites ............................................................................................................................... 3.2. Dye transport ..................................................................................................................................... 3.3. Fluorescent dyes combined with immunocytochemistry: general considerations .................................................. 3.3.1. Fixation ................................................................................................................................... 3.3.2. Mounting and Storage ................................................................................................................. 3.3.3. Fading ..................................................................................................................................... 3.3.4. Washout .................................................................................................................................. 3.35. Visualization ............................................................................................................................. 3.4. Fluorescent dyes combined with immunocytochemistry: specific considerations .................................................. 3.4.1. Fast Blue and True Blue ............................................................................................................... 3.4.2. Propidium Iodide ....................................................................................................................... 3.4.3. Primuline ................................................................................................................................. 3.4.4. Diamidino Yellow ...................................................................................................................... 3.4.5. Nuclear Yellow, Bisbenzamide and DAPI ........................................................................................ 3.5. Collateral tracing ................................................................................................................................. 3.5.1. Fast Blue (or True Blue) plus Diamidino Yellow ................................................................................. 3.5.2. Primuline plus Diamidino Yellow ................................................................................................... 3.6. Tracing of neurons with multiple putative transmitters ..................................................................................

103 103 104 104 104 104 105 105 105 106 106 112 112 113 115 115 I15 116 117

Present addresses: * Department of Anatomy, University of Bristol, Medical School, University Walk, Bristol, BS8 ITD, G.B.; ** Istituto di Clinica Dell Malattie Nervose e Mentali, Universita Cattolica del Sacro Cuore, Facolta di Medicina e Chirurgia, Largo Agostino Gemelli 8,00168, Rome, Italy. Correspondence: L. R. Skirboll, Chief, Electrophysiology Unit, Clinical Neuroscience Branch, NIMH, Bethesda, MD 20205, U.S.A.

100 3.6.1. Adjacent section method .............................................................................................................. 3.6.2. Elution restaining method ............................................................................................................ 3.7. Recommended procedures ....................................................................................................................

117 117 117

4. Discussion ...... ........................................................................................................................................ 4.1, General principles .............................................................................................................................. 4.2. Choice of fluorescent markers ................................................................................................................ 4.3. Collateral tracing ............................................................................................................................... 4.4. Tracing of neurons with multiple antigens ..................................................................... ........................... 4.5. Other studies with fluorescent tracers and immunofluorescence ..................................................................... 4.6. Other approaches _, ,. ., .___. __. _. .._._. ......... ......... 4.7. Final comments ._. ___.

171 133 123

5. Summary

1’3

_. ., ._.

Acknowledgements References

._. _..._,

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I I9 1IO I Ic) 130 I20

I23

._.

1. INTRODUCTION

During recent years, new anatomical techniques have been introduced which have opened up unique possibilities to trace nerve projections both in the central and peripheral nervous system. In particular, the use of radioactively labeled amino acids and horseradish peroxidase (HRP) as markers for anterograde and retrograde tracing of neuronal pathways have proved to be powerful tools?t,“‘.hY,71 (see also refs. 23,79, 105). Both anterograde autoradiographic and retrograde peroxidase tracing techniques make use of certain properties of neurons, i.e. the uptake of markers into the neuron, their incorporation into certain compartments and their axonal transport. Since these properties are, in principle, general to all neurons, these tracing techniques are in one sense ‘non-specific’. That is, they permit the mapping of the pathway, but provide no information regarding, for example, the specific neurotransmitter(s) present in the neurons. The last 10 years have also seen a remarkable increase in the number of putative transmitters described in the central nervous system (CNS). Thus, cholinergic and monoaminergic neurotransmission have been joined by a growing number of neuropeptide transmitter candidates. Extensive attempts have therefore been made to identify transmitters within neuron populations and a number of different strategies have been employed: the acetylcholinesterase (AChE) staining techniquesg; the formaldehyde fluorescence method for visualizing monoamines”4m’h;

123

autoradiography for studying GABA uptake site+,J7 and immunocytochemistry using antisera to a variety of substances, including catecholamine synthesizing enzymessgJY and many peptide+. In general, such transmitter-specific histochemical methods allow visualization of the transmitters and/or their synthetic enzymes in nerve endings, and often in cell bodies. Axons are, for the most part, difficult to identify in view of their low content of these substances. In any case, to follow axons along their entire course in serial or semiserial sections through the brain of even a small animal such as the rat is time-consuming, tedious and expensive. There is, therefore, a demand for a method by which projections neurons with identified transmitter can be traced. In previous studies, transmitter-specific tracing was successfully carried out in experiments on retrogradely transported HRP combined with AChE stainingd”,7x.“” and with tyrosine hydroxylase (TH) immunohistochemistry on the nigrostriatal dopamine system”7.73. Several other groups have also made similar attempts. partly by combining immunocytochemistry and HRPYX, but also formaldehyde-induced fluorescence and HRPX.12. and monoamine oxidase histochemistry and HRPxx. In the latter study, the correlation was not performed on the same sections. As early as 1970, it was recognized that certain dyes such as Evans Blue could be retrogradely transported in axons6l.r(lh. More recently, Kuypers and coworkers have systemically tested numerous dyes and introduced a number of agents which are well-suited for retrograde transport and which also fluoresce at a variety of emission wave length+sJ+hx. Subse-

101 quently,

several groups have described

neous use of these retrograde

rescence or formaldehyde-induced tochemistry for tracing precisely amine.

AChE

positive

the simulta-

and peptide

fluorescence hisidentified monopathways

in the

~~~1.2,9-11,13,14,18.49.51,52.80,87.89-91.97,101,102,1~,1~8,110.

as well as in the peripheral nervous system26.27.4i.83. Immunocytocyhemistry in combination 112-114.120

with these retrogradely

2. EXPERIMENTAL PROCEDURES

dyes and immunofluo-

transported

fluorescent

dyes

2.1. Dyes The dyes tested in experiments grade

tracing

on combined

and immunocytochemistry

retro-

were

se-

lected on the basis of extensive screening carried out by Kuypers and collaborator&5,63-6s. In Fig. 7 and in the Discussion

the excitation

and emission

of these dyes are given as published

spectra

by Skagerberg

offers the advantage that, in principle, it permits the visualization of any substance against which an anti-

and Bjorklund. Dann, Loewe

serum can be raised.

lowing dyes were tested. A: True Blue (code no. 150/129), (E)-2,Zvinylendi-benzofuran5-carboxamidin-diaceturate (injected in a 5% solution). Fast Blue (code no. 253/50), diamidino comB: pound chemically related to True Blue (3%). Nuclear Yellow (code no. Hoechst S 7691212c: (4-2)sulfamylphenyl)-6(6-(4-methyl-piperanzino)-2-benzimidazolyl)-benzimidaxol trishydrochloride (1%)74. Propidium Iodide (code no. P 5264; Sigma, S. D: Louis, MO, U.S.A.) (3%)53. Bisbenzimide (code no. Hoechst 33258) E:

Furthermore,

fluorescent

im-

munomarkers in combination with fluorescent retrograde probes permit the use of the same microscope in the analysis. Since the fluorescence characteristics of the retrograde dyes can, in several cases, be separated from fluorophores attached to the second antibody in the indirect immunofluorescence technique, these dyes allow visualization of a single labeled cell, and in some cases its dendritic arborization, and allow conjunctive and/or simultaneous identification of immunostaining in the same cell. In fact, retrograde tracing can be combined with elution-restaining immunocytochemistry, allowing studies on projections of neurons containing more than one transmitter candidate”i.97. Although we have studied fluorescent retrograde tracers and immunohistochemistry, it is important to note that other methods for combining retrograde tracing and immunohistochemistry have been developed. For information on this topic see Discussion and a recent reviewsa. In the present paper, we give a detailed description of a method by which neuronal pathways can be mapped and, at the same time, their chemical content characterized. The general applicability of this method was examined by using antisera raised to 3 categories of substances: a ‘classical’ small transmitter such as 5hydroxytryptamine (5HT), putative transmitter of peptide nature, thyrotropin releasing hormone (TRH) and substance P, and an enzyme involved in catecholamine synthesis, tyrosine hydroxylase (TH) in combination with several fluorescent dyes. Preliminary reports of this work have appeared and the methodology developed has been used in some studies4YJi.52.97.

F: G:

H:

Many of the dyes were obtained from and collaborators2s-s1,74J2. The fol-

(1%) 42.70.74.118. Primuline (code no. Eastman 1039; Eastman, Rochester, N.Y., U.S.A.) (10%). DAPI (code no. 102008 (18860); Serva, Heidelberg, F.R.G.) 2-(4-amidinophenyl)-indol6-carboxamidin-dihydrochloride (2.5%). Diamidino Yellow (code no. 288/26); diamidino compound, chemically related to DAPI (2%).

Further information on the drugs can be obtained from ref. 50. All compounds were dissolved or suspended in distilled water. True Blue and Nuclear Yellow were relatively insoluble and were therefore sonicated for 30 min after initial mixture, and sonication was repeated for at least 5 min before each use. Although Primuline and Propidium Iodide were not totally soluble, sonication was not necessary to dissolve them adequately for injection. In some cases, the dyes were stored at -20 “C over a several-month period prior to injection, but in no case did this seem to affect the fluorescence intensity or the retrograde transport of the dye.

102 2.2.

Injection procedures

and transport time

Male albino rats (150-200 taxically unilaterally

g) were injected

brains from both groups were processed stereo-

with dye either into the caudate

nucleus (A, 8380 pm; L, 3500 pm; V, 5000 pm according to the atlas of Konig and Klippela, the amygdaloid complex (A, 4890 pm; L, 4500 pm; V, 10,000 pm) or into the spinal cord approximately at the level of the C5 segment. jected by hand via a Hamilton 0.2-0.3

Dyes were either insyringe in a volume of

~1 or via a glass micropipette.

Pipettes were

pulled and broken back to a tip diameter of 25-50 pm, attached to a Medical Systems Pump (Great Neck, NY, U.S.A.) which permitted the delivery of small volumes of dye (0.1-0.2 ~1). In all types of administration, the instrument was held in place for a minimum of 3 min after injection was complete and raised slowly to reduce tissue damage and reflux movement of the dye up along the injection tract. After injection of the dye, animals were permitted to recover for specific periods of time to allow retrograde transport of the dye. Survival times ranged from 24 h to 10 days. Optimal survival times after dye injection have been described for many species by Kuypers and associates3-5.63-68. 2.3. Immunocytochemistry Immunocytochemical visualization of some peptides in cell bodies requires prior treatment of the rats with colchicine to arrest axonal transport and cause accumulation in the cell soma24.25.43. Therefore, the effect of such procedures on dye accumulation and transport were examined. In one series of experiments, colchicine (Sigma, St. Louis, MO) (6 pug/ml; total dose of 12Opg) was injected into the lateral ventricle immediately preceding injection of either Fast Blue or Propidium Iodide. Another set of animals received dye and were treated with colchicine 48 h later. Survival times of 24 h to 10 days were tested for each of the dyes examined. In all cases, rats were treated with colchicine 24 h and then processed as described earlier+46. Briefly after perfusion through the ascending aorta with 10% ice-cold formalin (40 g paraformaldehyde dissolved in 1000 ml of 0.1 M phosphate buffer according to Pease@) for 30 min, brains were dissected out and immersed in the formalin fixative for 90 min or 24 h and rinsed with 0.1 M phosphate buffer with 5% sucrose added 24 h later.

immunofluorescence laboratorsly. benzoquinone tion76.84.

according

for indirect

to Coons

and col-

A second fixative was also tested, P(0.25%) in a 5% formalin solu-

The brains and spinal cords were cut in a cryostat (Dittes, Heidelberg, F.R.G.) at a section thickness of 8, lo,14 or 20pm. Recently, we have used a warm plate (Dittes, Heidelberg, F.R.G.) inside the cryostat to dry the mounted sections before exposure to the air in the laboratory.

Injection

sites were cut and

examined. Brains injected with dye into the caudate nucleus and amygdaloid complex were sectioned at the levels of the substantia nigra and dorsal raphe, while sections from the medulla oblongata were taken to examine retrograde transport following dye administration into the lower cervical spinal cord. Sections were mounted on chromalun-gelatin coated glass object slides and the effects of mounting and storage on dye retention in labeled cells were examined. Mounting parameters included: unmounted sections; sections mounted in xylene. buffer, or oil. Sections were examined in the fluorescence microscope either immediately upon cutting, or after storing for l-24 h at room temperature or for 24 h at -20 “C. Sections were then processed for immunocytochemistry by first incubating with one of 4 antisera raised against: TH (dilution 1:200- 1:800)7’, 5-hydroxytryptamine (dilution 1:4OO)i”“. TRHr16 or substance Pl* at 4 “C for 24-48 h, rinsed in phosphatebuffered saline (PBS), incubated with either fluorescein isothiocyanate (FITC) conjugated swine antirabbit antibodies (DAKO, Copenhagen, Denmark) 1:lO or tetramethyl-rhodamine isothiocyanate (TRITC) (same as FITC) conjugated swine anti-rabbit antibodies (same supplier) as above for 30 min at 37 “C, rinsed in PBS and mounted in a mixture of glycerol and PBS (3: 1). 2.4. Visualization and evaluation Sections were examined in a Zeiss transmission fluorescence microscope equipped with an oil dark field condenser using the following filters (see Table I; for information on wavelengths at which dyes and immunomarkers are activated, see Fig. 7. taken from Skagerberg and BjorklundYS): True Blue and Fast Blue fluorescence were analyzed using a Schott UG-I filter for activation and a Zeiss 44 as a

103 secondary

filter as recommended

Skagerbergio

(Filter combination

by Bjorklund

and

to be distinguished

A). Propidium

Io-

one immunostain.

dide and Rhodamine (TRITC) fluorescence were examined using a Schott BP 546 as a primary filter and a

from each other, i.e. two dyes and

Since there are not 3 compounds can be distinguished

on spectral

which

was necessary

labeled cells, an additional stop filter, KP 560, was added to reduce shining through of the red dye. All

spectral characteristics. Excluding the dyes not suitable for combination with immunohistochemistry, as

remaining

discussed below, some dyes were selected, which had

dyes as well as FITC fluorescence

were ex-

plus an LP 455 filter) and a Zeiss 50 or LP 520 stop filter (sometimes plus KP 560 filter) (comb. C). Scopix RP-1 black and white film (Gevaert, Belgium), and Kodak High-speed Ektachrome color film (160 Tungsten, Eastman Kodak, Rochester, NY, U.S.A.) were used for photography. Elution experiments. In one series of experiments, sections were first incubated with the primary antiserum and processed according to the elution technique of Tramu et al.rOs as described previously51. Briefly after examination and photography of the retrograde dye, the sections were incubated with the primary antiserum and FITC- or TRITC-conjugated antibodies and photographed. The sections were then immersed in a mixture of potassium permanganate (KMnO,) and sulphuric acid (H,SO,) for 30-60 s. Sections were examined in the fluorescence microscope again and were, provided no fluorescence was observed, incubated with FITC-conjugated antibodies for 30 min at 37 “C and examined again. If fluorescence could not be observed, it was assumed that the first antiserum had been removed and the sections could then be processed for the second antigen. Antiserum to the second antigen was applied to the section, followed by FITC-conjugated antibodies as described above. The patterns of the second antigen were analyzed, photographed and compared with the distribution of that of the first one and of the retrogradely transported dye, all in the same section. 2.5. Studies on collaterals Several studies using fluorescent tracers have been carried out to trace collateral projections6.7.63,64,6*.111 and van der Kooy and Hattori combined collateral tracing with formaldehyde fluorescence histochemistryiw. To combine such studies with immunofluorescence histochemistry, 3 fluorescent compounds have

compartmentalization

in addition

a cellular localization plasmic

markers

the labeling

it

LP 590 as a secondary filter (comb. B). When FITC conjugated antibodies are used for Propidium Iodide

amined with a Schott BG 12 (3 or 4 mm) or preferably one or two KP 500 excitation filters (sometimes

to differentiate

available

characteristics,

different

studied

by cellular

to differences

in

from the diffuse cyto-

with immunohistochemis-

try: Primuline which has a distinct granular cytoplasmic localization, Fast Blue (or True Blue) which has a primarily cytoplasmic localization, and Diamidino Yellow which primarily is found in the nucleus. 3. RESULTS

3.1. Dye injection sites The analysis of the injection sites in the caudate nucleus revealed in most cases a central area in which the dye occurred in very high concentrations, surrounded by a zone of dye which projected concentrically away from the central dye deposit, in which individual cell bodies labeled with dye were observed (Fig. 1A). Ejection of the dye using the Hamilton syringe technique most often resulted in a central area of necrosis away from which dense staining could be observed (Fig. 1A). Injections of 0.2 ~1 of dye using this method gave rise to a site diameter of loo-150 pm (central necrosis plus intensely labeled circumference). This zone was surrounded by a layer of a more weakly labeled area. Dye injections made by application of micropressure from glass micropipettes reduced the central necrotic area as well as the amount of dye ejected at any one spot; these resulted in site diameters of less than 100 pm. The insolubility of True Blue, Nuclear Yellow and Diamidino Yellow limited their usefulness in the pressure ejection system: the dye tended to precipitate in the micropipette tip and clog the system. This required high pressure for ejection resulting in difficulties to deliver small amounts of dye. Fast Blue, Propidium Iodide, Primuline and Bisbenzimide, however, were easily ejected by either method of dye delivery. Preliminary evidence also suggests that Fast Blue can be ejected from a micropipette using iontophoretic negative currents (unpublished).

104 Subsequent

processing

munohistochemistry

of the same section for im-

with proper secondary

antibod-

ies allowed comparison of the localization of the injected dye with the distribution of, for example, nerve endings the potential

containing

a certain

transmitter,

i.e.

uptake sites of the dye. In Fig. lB, do-

pamine (TH containing)

nerve endings in the caudate

tems. Delays markedly

of more than 4 days did not lead to

brighter

fluorescence.

True Blue some labeling

but for Fast-

of neighboring

and

glial cells

could be observed 8-10 days after injections. Bisbenzimide, DAPI and Nuclear Yellow (Fig. 2E), but not Diamidino bodies

Yellow, moved rapidly out of the cell

and into neighboring

glial tissue.

Since.

in

nucleus are demonstrated in relation to the spread of Fast Blue injected at this site (Fig. 1A). Further-

most cases, it is necessary to follow a dye injection with a 24-h colchicine treatment, the use of these

more, the TH staining

dyes invariably

also gave direct information

on the extent of the necrotic area, since here the TH nerve endings had degenerated. 3.2. Dye transport The times required between dye injection and animal sacrifice to produce optimal retrograde transport and minimal non-specific labeling have been described by Kuypers and collaborators (for refs. see above). These times may vary from several hours to several days, and even weeks depending upon the dye, the species and the length of the pathway being traced. In studies on the effect of colchicine on transport of dye, injections were made into the caudate nucleus immediately followed by an injection of colchicine into the lateral ventricle; in a second group, dye was injected into the caudate nucleus, and colchicine 48 h later into the lateral ventricle. Virtually no labeled cells were observed in animals in which dye and colchicine were injected consecutively. In contrast, many intensely labeled nigral cells were observed in animals, in which colchicine was administered 48 h after the dye. In view of these findings, subsequent retrograde tracing in combination with immunocytochemistry required a two-step procedure, i.e. the animals were injected with the appropriate dye and allowed to recover for a period sufficient to permit retrograde transport (24-72 h or more), and then colchicine was injected into the lateral ventricle to arrest axonal transport of transmitter, peptide or related compound. Several antigens, for example TH, could, however, be easily demonstrated in cell bodies also without colchicine treatment. In studies of optimal transport time, it was found that True Blue, Propidium Iodide, Diamidino Yellow, and Primuline were all adequately transported over a 48-h period in both the nigro-striatal (Figs. lC-H, 3, 4) and bulbospinal (Figs. 5, 6) sys-

lead to a massive

rounding glial cells (and perhaps (Fig. 2E).

labeling adjacent

of surneurons)

Finally, with regard to dye transport, the effects of dye concentration on label accumulation (intensity) was evaluated. Propidium Iodide, Primuline, Fast Blue and True Blue were injected bilaterally into the caudate nucleus of a series of rats in concentrations of 3% and lo%, respectively. Forty-eight hours after dye injection, examination of dye intensity in cell bodies in the substantia nigra revealed that higher concentrations of dye did not label cells more efficiently, i.e. a greater volume of the cell labeled or with greater intensity than the lower concentrations. If these findings are generally valid, they must, however, be tested also in other systems. 3.3. Fluorescent

dyes combined

with immunocyto-

chemistry: general considerations

Since immunocytochemical procedures require that the tissue sections pass through incubation in water phase (antisera), followed by washing several times and exposure to several temperature changes, it was necessary to determine to what degree each phase affected the retention of the dyes. This was tested for True- and Fast Blue, Primuline, Propidium Iodide and Diamidino Yellow. The following factors were of importance. 3.3.1. Fixation. Fixation with ice-cold 10% formalin for 2 h was compatible with all dyes tested in the sense that it did not abolish the fluorescence or cause diffusion of the dye. It also resulted in retention of the antigens and their antigenicity to an acceptable degree. Fixation for 24 h did not, in our hands, result in improvements in any of the aspects mentioned above. Fixation with a formalin-PBQ mixture, in contrast, could not be used since none of the dyes exhibited fluorescence after this procedure. 3.3.2. Mounting and storage. Histological sections

105 are generally for

mounted

microscopic

Mounting

to provide optimal conditions

examination

dye-labeled

(the preferred

and

photography.

sections in glycerol and water

method

in immunocytochemistry)

of-

certain

extent dependent

mosphere.

A high humidity

on the humidity

ble to photograph cells, due to a combination of fading and diffusion. The use of a warm plate in the cryo-

ten enhanced movement of the dye out of the cell, reducing dye intensity and sometimes leading to label-

stat to dry the sections immediately and before exposure

to the atmosphere,

ing of neighboring

duced fading and/or

diffusion.

glia. This was not seen when

mounting in Entellan (Merck, Darmstadt, F.R.G.) or oil, but the use of these media made subsequent immunocytochemistry

difficult.

out that this dye diffusion

It should be pointed

was observed

with True

Blue, Fast Blue, and Diamidino Yellow; no dye extrusion was seen in cells labeled with Propidium Iodide or Primuline. For photographic purposes, the best mounting media for the 3 diffusible dyes proved to be xylene, although occasionally Fast Blue and True Blue-labeled sections showed diffusion which then could be enhanced by the immunoprocessing. Furthermore, in our experience, mounting in xylol prior to immunohistochemistry often resulted in a weaker immunofluorescence. We therefore often photographed sections without mounting medium and cover-slip. A further alternative is mounting in oil, but this procedure also influences immunohistochemistry in a negative way. Under some conditions it might be necessary to store dye-labeled sections prior to processing for immunocytochemistry. Such sections were best preserved by keeping them frozen and unmounted in a sealed container. If sections were stored mounted in buffered glycerol, there was an increased incidence of dye migration out of labeled cells (for True and Fast Blue and Diamidino Yellow). However, it should be pointed out that storage of unmounted sections did seem to lead to an increase in background fluorescence in sections subsequently treated for indirect immunocytochemistry. 3.3.3. Fading. A serious detriment to accurate counting of labeled cells came from fading and/or diffusion during exposure to ultraviolet (UV) light. This could be analyzed by exposing sections to UV light and comparing the number of labeled cells before and after various periods of timed exposure in the fluorescence microscopy (60 s-5 min). It was found that Fast Blue and particularly True Blue were susceptible to fading within a 120-s period. Propidium Iodide and Primuline, however, seemed resistant to fading under these conditions. The degree of fading was to a

in the at-

made it virtually impossi-

after sectioning

Blue that had diffused into the nucleus seemed fairly resistant to fading. 3.3.4.

greatly re-

Fast Blue and True

Washout. In order to determine

(see below), how immu-

nocytochemistry affected retrogradely labeled cells, adjacent brain sections were cut on a cryostat, the labeled cells were photographed and counted in one of the sections. The adjacent section was processed for immunohistochemistry, a photograph was taken after the completion of the immunocytochemical procedure and the number of remaining labeled cells was counted. By comparing the two photographs, it was found that True Blue and Fast Blue were subject to washout and a loss or marked decrease in intensity was observed in many cells (Fig. 2A, B) (see below). Propidium Iodide and Primuline, however, reliably remained in labeled cells. Prolonged fixation did not appear to enhance retention of Fast Blue or True Blue in cells. The degree of washout varied considerably between different systems and also between different cells. Thus, some cells were very resistant and were strongly fluorescent after the immunohistochemical processing, whereas other cells disappeared completely. With Fast Blue and True Blue there was often a diffusion of the dye into the nucleus with concomitant decrease in intensity in the cytoplasm (cf. Fig. 3A and D). 3.3.5. Visualization. The emission and activation maxima of each of the fluorescent dyes have been examined in detailto, (see Fig. 7). The emission characteristics of the dyes result in clear differences in fluorescence color of these substances. Under proper filter combinations, Propidium Iodide and TRITC (rhodamine) (a fluorophore conjugated to the second antibody) both fluoresce red or orange-red; True Blue and Fast Blue show a blue color; Bisbenzimide, Nuclear Yellow and Diamidino Yellow are whitishyellow; while FITC (another second antibody fluorophore) fluoresces green. In several cases, the emission and excitation characteristics of these dyes allow effective separation from some of the fluorophores mostly conjugated to the second antibody of the im-

106 TABLE

I

Filtercombinations System Blue (comb.

A)

Red (comb. B) Green (comb.

C)

Fluorophore

Zeiss microscope

Leitz microscope

Fast Blue True Blue Diamidino Yellow TRITC Propidium Iodide FITC Primuline Diamidino Yellow

Primary: Schott UG 1 Secondary: Zeiss 44

Filter system A

Primary: Schott B 546 Secondary: LP 590 Primary: KP 500 + LP 455 (Zeiss FITC 09; BP 450-490) Secondary: LP 520 + KP 560 (Zeiss FITC 10; BP 520-560)

Filter system N2

munocytochemical process, i.e. FITC and TRITP. Excitation of the same section at different wavelengths allows separation of dye labeling from immunofluorescent staining in some cases. The selection of appropriate microscopic filter combinations makes it possible to examine a single section and visualize the dye by using one filter combination and the immunofluorescent staining by using another (for details see below). Thus, the ability to differentiate between dye and immunofluorophore is dependent upon using the right combination of dye(s), conjugated second antibody and filter. For this reason, each of the dyes will be discussed with regard to their compatibility with the two routine immunofluorescence markers, FITC and TRITC. The combination of filters used are listed in Table I, where blue (comb. A), red (comb. B) and green filter combinations (comb. C) are described for both Zeiss and Leitz microscopes.

3.4. Fluorescent

Filter systems

K2 of I2

dyes combined with immunocyto-

chemistry: specific considerations 3.4.1. Fast Blue and True Blue. Forty-eight hours after injection of Fast Blue or True Blue into the caudate nucleus, labeled cells were observed in the substantia nigra (Figs. lC, D, 2A, 3A) and dorsal raphe nucleus of the rat. Injections of similar volumes and survival times into the cervical spinal cord followed by similar survival times resulted in labeled cells in a variety of brainstem sites such as the medullary raphe nuclei (Figs. 5A, C, 6A, C), the pons (Fig. 2C). the periaqueductal grey, as well as forebrain areas such as the cortex. True Blue and Fast Blue both gave fluorescence throughout the cytoplasm. Fast Blue fluorescence was often more intense and granular (Fig. 3A) in nature than the True Blue; however, both agents seemed to fill out the cell and dendritic processes

Fig. 1. A-H. Fluorescence (A, C, D, E) and immunofluorescence (B, F, G, H) micrographs of the caudate nucleus (A. B) and substantia nigra (C-H) after injection of Fast Blue into the caudate nucleus 48 h prior to sacrifice. A, B: the micrographs show the same section photographed before (A) and after processing for immunohistochemistry with antibodies to tyrosine hydroxylase (TH) (B). In A, the spread of Fast Blue around the injection site is demonstrated. Note central zone of necrosis (asterisk) surrounded by zones of intense labeling (arrow heads in A) and a zone of light labeling mainly of cell nuclei. The TH-positive nerve endings exhibit a dense network which is absent only in the zone of necrosis indicated by arrow heads in B. The immunostaining allows a more precise judgement of the extent of the necrotic zone indicated by absence of positive fibers. Note absence of staining in globus pallidus (gp). C. E. G: micrograph taken before immunohistochemistry showing retrogradely Fast Blue labeled cell bodies under blue filters (comb. A). In E, the same area is shown under green filter combination (comb. C). Note that all Fast Blue labeled cells more or less intensely shine through the green filters (comb. C) in green colour. G shows the same section after incubation with antiserum to TH under red filters (comb. B). Many Fast Blue labeled cells are TH-positive (arrows), but some TH-positive cells (double headed arrow) contain only TH and no dye. D, F, H: higher magnification of retrogradely Fast Blue labeled cells under blue filters (comb. A) (D). In F. the same area of the same section after incubation with TH antiserum is seen under red filters (comb. B) demonstrating double labeling of many cells (l-6). However, some cells contain only Fast Blue (asterisks), whereas others contain only TH (arrows). In H. a double exposure has been carried out, i.e. first exposure under red filters (comb. B) followed by exposure under blue filter (comb. A). The exposure time under the two filters will decide the relative intensity. In H, there is a slight overexposure under blue filter giving the whole photo a hue in blue. Bars indicate 50pm. A and B, C, E and Gas well as D, F and H. respectively. have the same magnification.

108

109 equally well. Both True Blue and Fast Blue were sus-

through

ceptible

peared

mounting

to diffusion

out of the cell in response

to

media such as glycerol and PBS and, to a

the blue filter system

(comb.

A) and ap-

here as a blue (Figs. lC, 3A), but a strong

did,

Fast Blue labeling but not True Blue could be seen through the green filters (comb. C), where it ap-

however, improve the clarity of micrographs particularly at high power magnification (X 25 objective).

peared green (Figs. lE, 3B) and could be difficult to distinguish from FITC-induced fluorescence. This

Exposure

cence from weakly labeled cells, and strongly reduce

shining through was markedly cases where immuno-processing

the fluorescence intensity in strongly labeled structures. Both True Blue and Fast Blue were also sus-

less pronounced disappearance of dye (Fig. 3C, D). Fast Blue and True Blue were only faintly visible

ceptible

through

certain

extent,

with xylene.

Xylene

mounting

time of 3 min could totally ‘erase’ fluores-

to washout

as a result of the immunocyto-

chemical procedure (Fig. 2A, B). Analysis of nigral cells in adjacent sections revealed that 32% + 4% and 33.5% + 4% of Fast Blue and True Blue-labeled cells, respectively, were no longer visible (i.e. dye labeled) or markedly decreased in fluorescence intensity after the section had undergone the repeated washes and staining requisite to immunostaining (Fig. 2A, B). The Blue dyes often diffused into the nucleus during immunohistochemical processing (Fig. 3D). This nuclear blue fluorescence then seemed fairly stable and resistant to fading by UVlight (Fig. lD, H). Fast Blue and True Blue were routinely viewed

the red filters (comb.

reduced or absent in resulted in more or

C) and this was true

only for the most intensely labeled neurons and could only be seen before immunostaining. After staining, the intensity of the dye was so much reduced (see below) that no fluorescence penetrated the red filter (Fig. 1G). Thus, when combined with immunocytochemistry, Fast Blue should be followed by TRITClabeled second antibodies. This allows both dye and TRITC to be viewed in the same section by alternating filters and reduces the possibility of false positives. Sequential exposure of Fast Blue fluorescence and TRITC-induced immunofluorescence then allows clear documentation of double stained cells (Fig. 1H). It should be noted that TRITC-stained

4 Fig. 2. A-E. Fluorescence (A, B, C, E) and immunofluorescence (D) micrographs of the substantia nigra (A, B, E) and lateral pons (C, D) after injection of Fast Blue (A-C) or Nuclear Yellow (E) into the caudate nucleus (A, B, E) or the spinal cord (C). A, B: the micrographs show the same section before (A) and after (B) exposure to UV-light for 2 min (blue filters, comb. A). Note marked decrease in fluorescence intensity in all cells as well as virtually complete disappearance of cell bodies (arrow) and processes (arrow head). C, D: the micrographs show the same section before (blue filter, comb. A) (C) and after processing for immunohistochemistry with antiserum to tyrosine hydroxylase (TH) and TRITC labeled second antibodies (red filter, comb. B) (D). Note that almost all cell bodies contain both Fast Blue and TH immunofluorescence, demonstrating that the catecholamine (noradrenaline) cell bodies of the A7 cell group project to the spinal cord. Asterisks denote the same blood vessels. E: micrograph shows a section taken 24 h after injection of Nuclear Yellow and 12 h after colchicine injection (green filter, comb. C). The section has not been processed for immunohistochemistry. Many cell bodies in the zona compacta (zc) are strongly labeled (arrows). Note marked in vivo diffusion into the zona reticulata, where many nuclei of probable glial cells are intensely fluorescent. Bar indicates 50pm. All micrographs have the same magnification.

Fig. 3. A-G. Fluorescence (A, B, D, E) and immunofluorescence (C, F, G) micrographs of the substantia nigra 48 h after injection of Fast Blue (A-D) and Propidium Iodide (E-G) into the caudate nucleus. A-D: the micrographs show the same area of the same section. A and B have been photographed under blue filters (comb. A) and green filters (comb. C), respectively, before immunoprocessing, whereas C shows distribution of tyrosine hydroxylase (TH) demonstrated with FITC-conjugated antibodies (green filters, comb. C). D shows these cells under blue filters (comb. A) after processing for immunohistochemistry. Note that Fast Blue shines through under green filters (compare e.g. l-3 in A and B). Several Fast Blue labeled cells also contain TH-like immunoreactivity (l-3). Cells a and b are Fast Blue labeled but seem to lack TH. x indicates a Fast Blue cell which possibly is TH-positive, but the weak immunostaining makes a decision difficult. z marks a TH-positive, Fast Blue negative cell body. D shows reduction in Fast Blue fluorescence after immunoprocessing (compare A and D) as well as a very weak shining through of green TH immunofluorescence as a weak green colour. Note that under these conditions the weak Fast Blue staining does not interfere to any major extent with FITC-induced immunostaining (compare D with C). E-G: the micrographs shows the same section demonstrating Propidium Iodide under red filters (comb. B) and FITC-induced TH immunofluorescence under routine green filters (C) (comb. C) and with additional stop filter KP560 to reduce shining through of red (C). Bar indicates 2.5pm. All micrographs have the same magnification.

110

111

112

cells can be viewed through

the blue filters (comb.

A), where they appear with a greenish-brown this hue can be clearly distinguished Blue or True Blue-labeled

color;

washings

revealed

that virtually

none of the cells were lost after immunostaining,

from the Fast

cells, when retrograde

cer and immunofluorescence

to immunochemical lowing concomitant

tra-

rescence in the same section.

marker have a different

sesses, however,

al-

analysis of dye and immunofluoPropidium

some disadvantages.

Iodide pos-

It does not ap-

cellular localization. If the two fluorophores (Fast or True Blue and TRITC) overlap, there will be a mixed

pear to be transported as effectively as some of the other dyes and it seems the most toxic of all the men-

appearance

tioned dyeseT. Propidium Iodide is routinely

and the intensity

of the respective

fluo-

rescence will decide what color will dominate. and therefore

red

filters (comb. B) and appears red. Under the routine

Since Fast Blue is subject both to fading and washout, it is possible to eliminate

viewed through

green filter combinations (comb. C), Propidium Iodide was shining through and appeared red or reddish-

separate,

at least to some extent, dyes from the section by extensive washing and/or prolonged UV exposure. If

yellow (Fig. 3F). Provided that the FITC-induced fluorescence was sufficiently strong, however, it was possible to ‘cover’ the Propidium Iodide fluorescence with FITC-induced immunofluorescence. By adding a Schott KP560 (‘red elimination’) filter, the intensity of the Propidium Iodide shining through could be further eliminated (Fig. 3G), although strongly dye-labeled cells still were visible. Propidium Iodide did not appear to diffuse from labeled cells upon mounting and did not fade in response to UV exposure or water solutions inherent to the immunocytochemical procedure. It was therefore not possible to wash out or fade away this dye prior to immunofluorescence. Thus, the separation of this fluorescent dye from the immunofluorophores was made by alternating between filter combinations and distinguishing the red fluorophore from the green immunostain. Since Propidium Iodide shone through the green filters with a red color, there was no risk for false positive results (c.f. blue dyes and green filters). 3.4.3. Primuline. Injection of Primuline into the caudate nucleus resulted in labeled cells in the sub-

successful, this allows combination also of Fast Blue with FITC-induced fluorescence (Fig. 3A-D). Thus, the Fast Blue labeled cells were first photographed and then repeatedly washed and/or exposed to UV light. After establishing that the dye fluorescence in fact has disappeared, the section could then be processed for immunohistochemistry. It may, however, be difficult or even impossible to extinguish strongly labeled Fast Blue cells. Sequential photography, i.e. before and after immunohistochemistry, as described above, may also be preferable if immunofluorescence TRITC labeling is very strong, since, as said above, the red dye then may mask a (weak) Fast or True Blue fluorescence. 3.4.2.Propidium Iodide. Injections of Propidium Iodide into the caudate nucleus resulted in labeled cells in the substantia nigra (Fig. 3E) and dorsal raphe. This dye filled the soma of the cell but not the nucleus; the dendritic tree was filled to a lesser extent than was observed with the Blue dyes. Quantitative evaluation of susceptibility of the dye -

Fig. 4. A-G. Fluorescence (A, C-E) and immunofluorescence (B, F, G) micrographs of the substantia nigra after injection of Diamidino Yellow plus Primuline into, respectively, the caudate nucleus and the amygdaloid complex (A-D) and into the caudate nucleus (E-G). A-D: numerous cells with a strongly bluish-white labeled nucleus and a weak blue cytoplasmic fluorescence can be seen under blue filters (comb. A) (A, C). After prolonged exposure to UV-light there is a decrease in the intensity of the nuclear staining but also an almost complete disappearance of cytoplasmic fluorescence (D). Instead, after this fading the cytoplasm contains numerous small granules which under the blue filters (comb. A) appears bluish-white. These granules appear yellow under green filters (comb. C) (not shown) and represent Primuline labeling. The nuclear stain as well as the blue diffuse cytoplasmic stain represent Diamidino Yellow. In B, the distribution of tyrosine hydroxylase (TH) is demonstrated under red filters (comb. B) using TRITC-conjugated second antibodies. Note that neither Diamidino Yellow nor Primuhne shines through the red filters (comb. B) under these conditions. Thus, the cytoplasmic Diamidino Yellow masks the Primuline granules unless UV-fading is induced. E, F: the micrographs show the same section. Strongly yellow Primuline labeled granules can been seen in numerous cells (l-6) under green filters (comb. C) and these granules do not shine through the red filters (comb. B), but only the red fluorescence of the TRITC conjugate can be seen (F). G: under green filters (comb. C) both the yellow Primuline labeled granules can be seen as well as the green FITC induced immunofluorescence representing TH. Note that some cells are double labeled (l-3), whereas other cell profiles (a, b) only seem to contain the immunostain. Bars indicate 25 pm. B has the same magnification as A, D as C and F as E.

113 stantia nigra (Fig. 4E, G) and dorsal raphe nucleus.

even strongly

The fluorescence

if the immunofluorescence

was solely

granular

and present

Primuline

cells were difficult to detect was strong. A possible so-

both in cell soma and large dendrites, and therefore the outline of the cell bodies was sometimes difficult to distinguish, especially when labeled cells were

lution is to use higher dilutions of the primary antibody, resulting in a weaker immunoreaction and an easier detection of Primuline labeling. Alternatively,

densely packed.

inspection

Primuline

was also resistant

to fad-

ing and diffusion as a result of exposure to UV light and mounting media, respectively, and thus permitted dye and immunostaining to be viewed in the same section in the same session without preliminary photography

of labeled cells.

Primuline could be viewed through either the blue filters (comb. A) or the green filters (comb. C), where it appeared as either bluish-yellow (Fig. 4D) or yellow (Fig. 4E, G), respectively. The granularity of the substance allowed it to be readily differentiated from immunofluorescence thus making a false positive unlikely. Since Primuline was also resistant to washout (see below), sections could be processed for immunostaning directly after cutting. Principally, both TRITC (Fig. 4F) and FITC (Fig. 4G) could be used as second antibody conjugates. The former, however, was preferred, since even a strong Primuline labeling appeared very weak under the red filter combinations (also when viewed before immunostaining). After incubation with TRITC-labeled antibodies, it was virtually impossible to detect the Primuline granules through the red filters (Fig. 4F). TRITC shone only faintly through the green filters (comb. C), and Primuline could therefore be examined in a single section using the green filters (comb. C) to view the granular dye and the red filters (comb. B) to view the immunofluorophore (c.f. Fig. 4E and F). If FITC-conjugated antibodies were used, the relative proportions of the intensity of Primuline and immunofluorescence labeling were important. Under favorable conditions, the results were good (Fig. 4G). However, if the cells were strongly Primuline labeled and weakly immunofluorescent, it could be difficult to establish with certainty the presence of the immunomarker. If weakly Primuline cells were strongly immunopositive, it was difficult to distinguish the Primuline-labeled granules. Extensive UV exposure could then be used to ‘fade away’ the immunofluorescence to disclose Primuline, but this situation was clearly less suitable when numerous sections had to be ‘scanned’ for double labeled cells. Thus,

(and photography)

of Primuline-labeled

cells could be carried out before immunohistochemistry. Primuline

was associated

since the fluorescence

with some drawbacks,

was sometimes

difficult to dis-

tinguish from autofluorescence, particularly in weakly tracer-labeled cells, which made identification difficult and scanning of sections time-consuming. If young rats were used this did not represent a problem. 3.4.4. Diamidino Yellow. After injection of Diamidino Yellow into the caudate nucleus and spinal cord, labeled cells were found in the corresponding cell body areas, i.e. the substantia nigra (Fig. 4A, C, D) and nucleus raphe dorsalis and the lower brainstem, respectively. The cells had a strong fluorescence with the highest intensity in the nucleus, but often a weak fluorescence could also be observed in the cytoplasm, extending into the proximal dendrites. No evidence for diffusion out of cells and uptake into glial cells was obtained, in contrast to results obtained with Nuclear Yellow (see below). Diamidino Yellow had a tendency to diffusion during the immunofluorescence procedure. Thus, additional labeled cell nuclei could be seen after the immunofluorescence procedure. These newly labeled cells exhibited a clear gradient extending from areas containing strongly labeled cells of glial nature. It could not be excluded that some neurons were also labeled. Their fluorescence was very weak as compared to the originally marked cells. Under high atmospheric humidity, diffusion already occurred immediately after the cutting procedure and therefore no water soluble mounting media could be used. However, if dried on the warm plate in the cryostat or exposed to a stream of warm air and subsequent mounting in xylene, these problems could at least in part be overcome. Diamidino Yellow was visualized with the blue (comb. A) (Fig. 4A, C, D) and green filters (comb. C), where it had, respectively, a whitish-blue, or yellow or yellow-green fluorescence. It was also seen as red through the red filters, although only very weak-

114

115 ly. The incubation decreased

procedure

Diamidino

or UV radiation

Yellow-induced

only

fluorescence

intensity to a small extent. A weak cytoplasmic fluorescence, however, tended to disappear during incubation; this proved to be an advantage, since it allowed separation

of dye and immunostain

by cellular

Labeled neurons were mostly surrounded by fluorescent glial cells. The fluorescence partly disappeared after processing

3.5. Collateral tracings 3.5.1. Fast Blue (or True Blue) plus Diamidino Yellow. After the injection

compartmentalization. Against this background, the best immunofluorescence marker for combining with

of the caudate

Diamidino

the central

Yellow was TRITC

(Fig. 4B), but FITC

was also useful, since it could be distinguished through its cytoplasmic (i.e. transmitter-related) localization.

In many cases, however,

small cells, the immunofluorescence

particularly

with

occupied the cy-

toplasm as well as the nucleus. Thus, in reality a separation between dye and immunostain was not always as clear as might be expected on theoretical grounds. The ‘shining through’ of Diamidino Yellow through red filters, as seen by eye, was dependent on the intensity of the TRITC labeling. Thus, if the cytoplasmic immunofluorescent marker was weak, the nucleus appeared to have strong reddish Diamidino Yellow-induced fluorescence. On the other hand, if a strong immunofluorescence was studied, the nucleus did not appear to be labeled at all (Fig. 4B). 3.4.5. Nuclear Yellow, Bisbenzimide and DAPI. After injection of these dyes into the caudate nucleus, labeled cells were found in the substantia nigra and nucleus raphe dorsalis. Nuclear Yellow and Bisbenzimide gave a yellow fluorescence in the nucleus as well as the cytoplasm, although Bisbenzimide fluorescence was considerably stronger than that seen with Nuclear Yellow. Twenty-four but not 6 h after injection, a marked labeling of surrounding glia cells was seen with Nuclear Yellow (Fig. 2F). Both dyes were also subject to fading upon exposure to UV light and were completely washed out by the immunocytochemical procedure. DAPI gave a strong blue nuclear fluorescence and also labeled the cytoplasm.

for immunohistochemistry.

nucleus

amygdaloid

of Fast Blue into the head

and Diamidino nucleus,

using

Yellow into blue filters

(comb. A), some cells in the zona compacta blue fluorescent cytoplasm and a yellow-white us. Adjacent

cells exhibited

a blue cytoplasmic

had a nuclefluo-

rescence, sometimes together with a blue fluorescent nucleus, whereas other cells had a strongly fluorescent yellow-white nucleus and a weakly fluorescent cytoplasm with the same color. These findings are in agreement with Kuypers et al. (1980) for Fast Blue and Nuclear Yellow, who described that these two dyes can be separated on the basis both of cellular localization and by their different colors at identical excitation wavelengths. The former criterion is, however, relative, since also Fast Blue tended to label the nucleus and since Diamidino Yellow also could label cytoplasm. The cytoplasmic labeling by Diamidino Yellow was, however, very weak and in practice did not interfere with the Fast Blue fluorescence. Similarly, the strong Diamidino Yellow nuclear fluorescence could be easily distinguished in the microscope, since it seemed to ‘override’ a possible nuclear Fast Blue fluorescence. After processing the sections with TH antibodies and TRITC-conjugated second antibodies, the cytoplasm of nigral dopamine cells was strongly red fluorescent. A weak nuclear staining was also observed representing Diamidino Yellow shining through. By switching filters, Diamidino Yellow appeared yellow-white and was almost as strong as before immunofluorescence processing. In several cells Fast Blue

Fig. 5. A-D. Fluorescence (A, C) and immunofluorescence (B, D) micrographs of the nucleus raphe obscurus of the medulla oblongata after injection of Fast Blue into the spinal cord and processing of two adjacent sections (A, C) (thickness 10pm) for indirect immunofluorescence using antibodies to TRH (B) and 5-hydroxytryptamine (5-HT) (D) and TRITC conjugated second antibodies. Comparison of A and C indicates that many Fast Blue labeled cell bodies appear in both sections (compare, for example, pairs of cell bodies labeled with coded arrows). Many retrogradely labeled cells contain TRH (compare B with A) and 5-HT (compare D with C). There are also examples of retrogradely labeled cells which neither contain TRH nor 5-HT (compare single headed and x-marked arrows in A and C with B and D). Many retrogradely labeled cells seem to contain either TRH or 5-HT (compare single- and double-crossed arrows in A-D). Some retrogradely labeled cells contain both TRH and 5-HT-like immunoreactivities (compare 3-crossed and 2-, 3-and 4-headed arrows in A-D). Bar indicates 50pm. All micrographs have the same magnification.

116 had been retained,

but the fluorescence

was much

weaker than before immunofluorescence processing as described above. In agreement with the results above, the relative

strength

of the labeling

with the

Blue dyes and Diamidino

Yellow, repsectively,

result in some difficulties

in evaluating

could

the results uf-

3.5.2. Primuline plus Diamidino Yellow. After injection of Primuline and Diamidino Yellow into the amygdaloid complex and caudate nucleus, respectively, cells in the substantia nigra exhibited a mixed appearance.

Some contained

granular

cytoplasmic

yellow (green filters, comb. C) or bluish (blue filters,

fer immunohistochemistry. Thus, a strong labeling with the Blue dyes and a weak one with Diamidino

comb. A) fluorescence. Other nous whitish blue fluorescence

Yellow could result in diffusion

A), which was strong in the nucleus and weaker in the

nucleus and difficulties

of the Blue dye into

to distinguish

low. In the reversed situation, could diffuse into the cytoplasm

Diamidino

Yel-

Diamidino Yellow and interfere with

the cytoplasmic Blue dye. Photography before immunohistochemistry was of great help under such conditions .

Fig. 6. A-D. These micrographs represent higher magnifications Fig. 5A. The same symbols have been used to indicate cell profiles cates SOpm. All micrographs have the same magnification

cells had a homoge(blue filters, comb.

cytoplasm (Fig. 4A, C). In the latter cells, the occurrence of Diamidino Yellow in the cytoplasm resulted in problems of identification of the cytoplasmic marker (Primuline), since this dye disguised the Primuline-labeled granules (Fig. 4C). This could be overcome by prolonged UV exposure resulting in

of parts of Fig. 5A-D roughly as indicated by the rectangle in as in Fig. SA-D. We refer to legend to Fig. 5 for details. Bar indi-

117 fading of the cytoplasmic pearance

Diamidino

of Primuline-labeled

Yellow and ap-

granules

in some cells

(Fig. 4D), whereas other cells seemed to lack such granules. After processing for immunocytochemistry, it was found that many dye-labeled cells also exhibited red TRITC-induced TH immunofluorescence

ous blue fluorescent raphed. Incubation TRITC-labeled

antibodies

several cells, this could also be seen by simultaneous examination

Yellow-labeled

After photography

identified

TH could

be

in the same section.

with blue and red filter combinations.

HT antiserum,

of 5-HT staining

with anti-

P and TRITC-conjugated

3.6. Tracing of neurons with multiple putative trans-

bodies. Some cells contained

mitter As discussed previously48, two main approaches may be used to identify several antigens in a cell: (1) staining of thin adjacent sections with different antisera; and (2) elution of the first antiserum after photography and restaining with a second antise-

noreactivity

With the former approach, it is only necessary to be able to distinguish one tracer and one immunostain and the same combination can be used for both adjacent sections. With the second method the type of elution technique will be important. In the present study only elution according to Tramu et al.107 was evaluated. 3.6.1. Adjacent section method. After injection of Fast Blue into the spinal cord, numerous cells in i.a. the raphe nuclei of the medulla oblongata were labeled (Figs. 5A, C, 6A, C). When adjacent sections were analysed, it could be observed that profiles of the same labeled cell sometimes appeared in two sections (Figs. .5A, C, 6A, C). After photography and incubation of adjacent sections with antiserum to TRH (Figs. 5B, 6B) and S-HT (Figs. 5D, 6D), respectively, followed by TRITC-labeled second antibodies, many cells containing both Fast Blue and immunostaining were observed, some of them containing two of the antigens studied (Figs. 5,6). 3.6.2. El&ion-restaining method. After injection of the various tracers into the caudate nucleus, cell bodies in the substantia nigra were strongly fluorescent. Treatment of the sections with KMnO, as eluentr07 resulted in a complete disappearance of all dyes tested, i.e. Fast Blue, True Blue, Propidium Iodide, Primuline and Diamidino Yellow. Fast Blue was injected into the spinal cord. Sections from the medulla oblongata contained numer-

and elution of 5-

the sections were incubated

serum to substance

rum82,107,115.

resulted in many cell bod-

ies e.g. in the medullary raphe nuclei containing both Fast Blue and 5-HT as revealed by comparison of micrographs. Since Fast Blue was in part retained in

(Fig. 4B). By switching between red and blue (or green) filter combinations, Primuline plus Diamidino cells also containing

cell bodies, which were photogwith antisera to 5-HT followed by

in addition

substance

anti-

P-like immu-

to Fast Blue and 5-HT.

3.7. Recommended procedures Of the compounds tested, Fast Blue, True Blue, Propidium Iodide, and to a lesser extent Primuline and Diamidino Yellow were the ones best suited for combination with immunocytochemistry, whereas Bisbenzimide, Nuclear Yellow and DAPI were not compatible with this type of histochemistry. In the choice between Fast Blue and True Blue, the following considerations could be made. True Blue can be combined both with FITC- and TRITC-labeled second antibodies, Fast Blue preferably with TRITCconjugated ones. Fast Blue is easier to dissolve than True Blue. True Blue may diffuse less from the site of deposit and may therefore be a more precise marker and may perhaps be taken up into nerve endings for a longer period. 1. Dyes should be injected either using a 1 or 5 ~1 Hamilton syringe or a glass micropipette attached to a pressure ejection unit. Efforts should be made to limit the volume of dye injected to reduce the spread to adjacent structures not associated with the target neuronal population. The injection instrument should be left in place for at least 5-10 min after the completion of dye injection to reduce reflux of the dye up the injection tract. 2. For all recommended dyes, a 48-72-h survival time is sufficient to permit optimal dye transport for both the projections studied here in the rat (caudate to nigra and lower cervical cord to lower brainstem). This may vary, however, between species and depending upon the length of the pathway being traced. 3. In the case of immunocytochemical identification of substances present in low concentrations in cell

118 300

350

400

)-W-m

_--_

I

500

600

--I -I

_-_--_---_k----_-_-_

450

Bb -e-w----

-w--m-

CA

i

DY

i

DAPI EE FB me_-

FITC

-I

GB NY Pf PI TRITC

m-m

5 HT TB I

600 Excitation wavelength

bud

Fig. 7. The intensity of the different emission peaks of the mercury lamp is indicated in the range between 300 and 600 pm. The horizontal bars indicate the wavelength at which the tracer compounds (right column) are efficiently excited. The dashed part of the bar indicates the range at which the compound emits 90-100% of its maximum fluorescence. The arrows mark for each compound the mercury lamp peak which gives optimum excitation in the fluorescence microscope (from ref. 95).

(such as is the case with many of the neuropeptides), rhe animal receives an intraventricular colchicine injection (60-120 pg in 20 ~1 for rats) 24 h prior to sacrifice. Colchicine treatment must bodies

follow transport of the dye and can not be administered on the same day as the dye is injected. 4. Animals, which should be young to avoid confusion with autofluorescent granules (Primuline injections), are perfused through the ascending aorta with 10% ice-cold formalin (40 g paraformaldehyde dissolved in 1000 ml of 0.1 M phosphate buffer according to Pease, 1962, essentially as described by HGkfelt et al. 1973) for 30 min. The brains are then dissected out, immersed in the same fixative for 90 min and rinsed in 0.1 M phos-

phate buffered 5% sucrose. After at least a 24-h rinsing, the brains can be cut in a cryostat (section thickness set at 10-15 pm), and, if available, put on a warm plate in the cryostat for 30 s. For Fast Blue and True Blue, and preferably also for Diamidino Yellow, especially if combined with the Blue dyes, sections should be immediately viewed unmounted under the microscope and photographed. In some cases, especially for optimal micrographs at higher magnifications, it may be necessary to briefly mount the sections with xylene. After photography, the sections can be processed for immunocytochemistry. For Primuline and Propidium Iodide, the sections can be taken directly from the cryostat and processed for immu-

119 nocytochemistry,

since neither

of these dyes are

from its cellular location

during immunohistochemis-

subject to wash out from the immunocytochemic-

try. It is a tedious procedure,

al procedure.

retrogradely

Since Primuline

may be disguised

by the immunofluorescence, it may, however, be advantageous to analyze and perhaps photograph for the indirect immunoof Coons and collabora-

tor@. Briefly, the sections are incubated appropriate

antiserum

with the

at 4 “C for 24-48 h, rinsed

in PBS, incubated with FITC (for use with Propidium Iodide or True Blue) or TRITC (for use with Primuline, Diamidino Yellow, Fast Blue or True Blue) conjugated antibodies for 30 min at 37 “C, rinsed in PBS, mounted in a mixture of glycerol and PBS (3: 1) and examined in a fluorescence microscope with an oil dark field condenser. 6. For visualization of dyes and immunofluorophores in a Zeiss or Leitz microscope, the filters listed in Table I are best employed. Scopix RPl black and white film (Gevaert, Belgium) or Tri-X ASA 400 Kodak (Rochester, NY) black and white films are used for all 3 dyes. Tri-X seems advantageous when photographing red fluorescence (TRITC, Propidium Iodide). For color photography, Kodak High Speed Ektachrome (160 Tungsten, Eastman Kodak, Rochester, NY) can be used. 4. DISCUSSION

4.1. General principles Retrograde tracing combined

since the recording

with immunohisto-

chemistry presents specific problems as compared to ‘conventional’ retrograde tracing, the main issue being that the section has to pass through histochemical procedures involving a water phase. This is in contrast with combination with aldehyde-induced fluorescence in freeze-dried tissue, since no diffusion problem exists under these circumstance@. When the sections have to be carried through a water phase, two options are available. First, the two steps can be ‘separated’, i.e. the retrograde tracing experiment is carried out, the results are recorded by photography and the sections are then processed for histochemistry and the ‘new’ staining patterns are photographed again and the results compared. This approach is taken, if the retrograde dye diffuses away

of

cells is made ‘blind’, i.e. many

retrogradely labeled cells will be photographed, which later may turn out not to contain any of the antigens explored with immunohistochemistry.

the sections before immunohistochemistry. 5. Sections are processed fluorescence procedure

labeled

proach offers some advantages. eluted completely,

Provided

The apthe dye is

there will be no problems

rating and distinguishing

between

ported dye and immunofluorescence

retrogradely

in sepatrans-

marker (see be-

low). The second alternative is to select a dye that is retained in the cells during immunohistochemistry. In this case, the dye has to have characteristics which allow it to be distinguished from the immunofluorescence marker. This may be achieved, if the retrograde- and immunofluorescence markers have different excitation and/or emission fluorescence characteristics, or if the dye is retained in a cellular compartment different from the immunostain. A third issue to be raised is that most animals have to be treated with colchicine 24 h before sacrifice, which causes accumulation of peptides and transmitters in cell bodies in sufficient amounts to allow detection by immunohistochemistry. Thus, it is a prerequisite that in vivo the dye does not diffuse out of the cell for at least 36-48 h, i.e. sufficient time for transport of the dye plus the colchicine treatment. It has been shown in this paper that colchicine blocks the retrograde transport of dye and thus colchicine can only be given after transport of dye is complete. Our initial attempts were based on the use of HRP as retrograde tracer combined with indirect immunofluorescence’s. However, several problems were encountered, since the sections had to be analysed in two different microscopes (light and fluorescence microscopes) and since HRP was sensitive to UV light. The introduction of fluorescent retrogradely transported markers offered a new approach. On the basis of the work of Kuypers and collaborator+s@-68, we tested a number of fluorescent dyes for their suitability for combination with immunohistochemistry. 4.2. Choice offluorescent markers Several dyes could be ruled out as this type of study, on several grounds: Nuclear Yellow and DAPI showed staining, representing transfer of dye

unsuitable for Bisbenzimide, marked glial from neurons

120 to glia, a process markedly enhanced by immunohistochemistry. This occurs after short times and even before

immunohistochemical

processing

makes

it difficult

with 24-h colchicine

treatment.

to combine

In the case of Primuline,

and

thus

the yellow gran-

ent study that retrograde tracing using Fast Blue, True Blue, Propidium Iodide, Diamidino Yellow or Primuline

can be successfully

combined

immunofluorescence

histochemistry

transmitter-identified

pathways.

with indirect

for mapping

of

This is in agreement

ular fluorescence of the dye was sometimes difficult to distinguish from autofluorescent granules. This

with several recent studies carried out in the CNS in our own and in other laboratoriesl.2,9-11,13~14.ls.49,~1,52.

was particularly

80,87.89-91,97,101,102.104,108,110,112-114,120,

evident in older rats and when study-

ing systems which do not transport large amounts of the dye. Furthermore, weakly Primuline-labeled cells were difficult to detect, making fast scanning extensive

areas and of many sections

of

at low power

magnification a difficult procedure. Also, after processing for immunocytochemistry with FITC-conjugated antibodies, the immunofluorescence may disguise the dye. For this reason TRITC proved to be a better immunofluorescence marker in combination studies with Primuline. True Blue and Fast Blue have similar qualities. They are effectively transported and give an intense fluorescence. True Blue is difficult to dissolve but may diffuse less from the injection site and be available for uptake and subsequent transport for a longer time. Both do, however, to a certain extent diffuse out of the cells during immunofluorescence processing and are subject to fading upon UV illumination. Diamidino Yellow is transported well but is subject to diffusion during the immunocytochemical procedure resulting in glial labeling. It should be emphasized, however, that problems exist even with the most favorable dyes. Thus, Fast Blue and True Blue are to a certain extent eluted during immunohistochemical processing. Since the degree to which this occurs is not predictable and varies between different systems and, from time to time, photographic recording should be carried out before immunohistochemistry. Propidium Iodide, which is resistant to fading and does not diffuse out of the cells during histochemistry, is not as well transported as, for example, Fast Blue67, and may therefore not be useful in all systems. It should be noted that in the present study favourable systems have been selected and that it may be more difficult to use the approach described here on other projections, for example neurons with small projection fields and neurons containing compounds which are more difficult to visualize than the antigens studied here. In spite of these draw-backs it is shown in the pres-

The best choice of dye is, in our experience,

Fast

Blue or True Blue and Propidium Iodide with a blue and red fluorescence, respectively. If TRITC- and FITC-conjugated second antibodies are used for the immunohistochemical part in combination with the Blue dyes and with Propidium Iodide, respectively, it is possible, by switching between appropriate filter combinations, to unequivocally distinguish between fluorescence induced by retrogradely transported dye and immunofluorescence marker. Thus, it can be decided whether or not a neuron containing a certain transmitter or peptide projects to (or through) the site of the dye injection. 4.3. Collateral tracing As convincingly shown in several papers, the retrogradely transported dyes can also be used to study collateral organization6,7,63.64,6s~lll, For example, Van der Kooy et al.111, using the red fluorescing Evans Blue and a mixture of DAPI and Primuline, giving blue fluorescence, could demonstrate double labeled cells in the mammillary body after injecting the dyes into thalamic areas and into the mesencephalit tegmental area, respectively. Other dye combinations have been, for example, Nuclear Yellow or Bisbenzimide, on one hand, and Fast Blue or True Blue, on the other hand6@JJY. Fluorescent dyes (DAPI) have also been combined with HRP for this purposet21. Also this type of study, i.e. using two retrogradely transported dyes, can be combined with immunohistochemistry. It could be shown that Diamidino Yellow can be used in combination with Propidium Iodide (FITC) or Fast Blue/True Blue (TRITC) or with Primuline (TRITC), since the dyes preferentially label different cellular compartments, nucleus and cytoplasm, respectively. 4.4. Tracing of neurons with multiple antigens The present study demonstrated that retrograde tracing can be combined with demonstration of mul-

121 tiple antigens

in the same cell. Two approaches

devised, (1) staining of adjacent secutive staining the first antibody.

were

sections and (2) con-

of the same section after elution The latter technique,

employed

all fluorescent

ied here were destroyed approach

by the KMnO,

was connected

tions: (1) photography

retrograde

be made at the latest before incubation

to their findings,

to Fast

True Blue, in fact,

resulted in a higher percentage

in

in the paraventricular-spinal system, as compared to Bisbenzimide (58%), HRP (24%) or HRP-polyacrylamide (39%). have arrived

elution,

none of the fluorescent

this

condi-

tracer has to with antise-

rum to the second antigen. As discussed above, we mostly prefer to photograph the dye before all immunohistochemistry to avoid problems with washout. (2) Since the first antibody is eluted, there is no need to distinguish between the immunomarkers for the first and second primary antisera. Since the fluorescent dye is destroyed, it can not confuse identification of the second immunomarker. Therefore, the separation problem is confined to the distinction between retrograde tracer and the fluorescent probe for the first antiserum. With regard to the first approach (the adjacent section method), also here the only requirement is being able to distinguish between retrograde tracer and one immunofluorescence marker. The choice of method depends on, for example, the antigens studied. If 5-HT and TRH have to be investigated, the adjacent section method has to be employed, since according to our experience KMnO, elution destroys both compound@, and thus makes it impossible to restain either of them. It is, however, important to note that other elution methods are available**Jis and that it may be possible to employ them in studies on retrograde tracing of neurons with multiple antigens. In conclusion, retrograde tracing combined with analysis of multiple antigens can be carried out according to the general principles outlined for combining one retrograde tracer and one immunofluorescence marker. 4.5. Other studies withfluorescent tracers and immunofluorescence A methodological study of combined fluorescence retrograde transport and immunohistochemistry has been published during the course of this investigation by Sawchenko and Swansor+@. They analysed several general parameters related to retrograde transport. Their dye of choice was True Blue, which, as de-

(88%) of labeled cells

Other groups studying

tracers stud-

with the following

of the retrograde

Blue. According

of

earlier studie@,97, was based on elution with acid KMnO, according to Tramu et al.107. Since, as shown in this paper,

scribed above, has very similar characteristics

at different

conclusions,

other systems showing

that

dyes in this respect is superior

to HRP54. We have not addressed

this question

in the

present study. With the exception of Propidium Iodide and Primuline, all dyes to a certain extent were affected by the immunohistochemical

procedure,

which caused

diffusion of the dyes and a reduction in the number of stained cells. Our findings on the nigro-striatal system revealed that about 113 of the Fast Blue cells were more or less washed out. Sawchenko and Swansons9 reported a 5% loss and van der Kooy and Sawchenkoil* a 15% loss with True Blue in their system. The latter authors also described loss of about 20% of Propidium Iodide-labeled cells. In a previous study, we observed an almost complete washout of True Blue in a bulbospinal enkephalin system52. According to Sawchenko and Swanson@ their fixation method is responsible for the better retention. In our experience, the retention varies considerably from system to system and is dependent particularly on the intensity of the labeling and/or perhaps on the cellular compartment, in which the dye is localized. With strong labeling, Fast Blue occupies a granular storage site (lysosomes?) and these cells are more resistant to washout, whereas diffuse cytoplasmic dye seems less resistant. With regard to the different fixatives, it may be pointed out that the formalin solution used by us is made up from 4% paraformaldehyde, which results in a 10% formalin solution, i.e. the same concentration as that used by Sawchenko and Swansor+. The difference may therefore be dependent rather on the length of the fixation time than on the fixation solution per se. Our reason for using shorter fixation times is that, in our hands, this seems to be preferable for ‘good’ immunohistochemistry. On the other hand, if systems are studied with very potent antibodies, one can easily fix for longer periods and obtain good immunohistochemical results. It may therefore be advisable to check every individual system and fix the brains as long as possible for a good immunoreaction, since this seems to favor re-

122 tention of the dye. Our recommendation

is, however,

using markers

when using Fast Blue (or True Blue), to photograph

certain

before immunohistochemistry.

transport,

Even a 5% loss as de-

nerve

Jacobowitz

prefer to use TRITC instead of FITC as fluorescence marker, since there is absolutely no ‘shining through’

(DBH)

dide, although

this drug does not seem to transport

well in all systems67. If the labeling may, in addition, 4.6.

is very strong,

it

‘shine through’ the FITC filters.

Other approaches

Although several papers have appeared using principally the same approach as the one outlined in ,~~~y18.26.27,41,4Y,51,52,59.83.89-91,Y7,10l,102.110,112-l14 this there are several other options available today for tracing transmitter-identified neuron projections (see ref. 50). A similar approach, i.e. using fluorescent dyes, was taken by several groups1a~tt.Ys.114but they combined the dyes with aldehyde-induced fluorescence. As mentioned above, the use of freeze-dried tissue circumvents the problem of diffusion of the dye, but requires that retrograde dye and fluorescent dye can be separated either by filters or by a differential cellular localization. These approaches are, however, limited to studies of monoamine neurons. Several groups have used HRP as a retrograde marker in combination with aldehyde induced fluorescence8J-14.32.75 or with monoamine oxidase histochemistryss. An alternative is HRP retrograde tracing combined with immunohistochemistry. Several groups have used HRP as retrograde tracer combined with immunofluorescence7398 or with PAP immunocytochemistryl~-l7.72.s6.s7.9Y~l21. Lechan et al.‘2 have demonstrated that the technique of retrograde transport of wheat germ agglutininY3,ra0 can be combined with immunofluorescence histochemical visualization of TH. Retrogradely transported fluorescent dyes have been combined with acetylcholinesterase (AChE) stainingl.9.120. A similar approach using HRP as retrograde tracer was earlier carried out by Warrrr’, Hardy et al. 40 and Mesulam7s. Retrograde transmitter-specific tracing can be carried out in principally different ways, for example by

and which,

can be detected

scribed by Sawchenko and Swansonsy represents a substantial basis for error. When using Fast Blue, we

of the dye with proper filter combination. If washing out has to be avoided, we recommend Propidium Io-

which are specifically endings

in the cell bodies.

et al.56 demonstrated

line synthesizing

enzyme

can be taken

taken

up into

after retrograde Thus,

that the noradrena-

dopamine

P-hydroxylase

up in sympathetic

elements.

Subsequently, it could be shown that this property of DBH can be used as a basis for retrograde tracing37.94,*rs.t2*.Several transmitters exhibit a specific re-uptake mechanism (see ref. 55), which can be utilized for autoradiographic identification (see ref. 47). It has been shown also that these compounds

are ret-

rogradely transported allowing transmitter specific identification of the cells of origin (see ref. 23). 4.7. Final comment With regard to choice between various combined methods, it is apparent that all techniques have advantages and disadvantages, and no one is perfect. There will in many cases be problems of balance to distinguish between retrograde tracer(s) and histochemical marker(s) for the transmitters in order to obtain clear-cut results. Sawchenko and Swansonss have pointed out some of the shortcomings of using fluorescent dyes, and we agree on several of these issues: fading and washing out of Fast and True Blue and uptake of True Blue into fibers of passage, an issue not tested by us for Fast Blue or any other dye. The possibility of transfer of dye between axons has been discussed and, on the basis of experiments by Kuypers and collaborators66~67.‘*~. such a process seems less likely to occur (see ref. 89). We would therefore, at present, prefer not to take any stand as to recommend a particular combined retrograde tracing-immunohistochemical procedure. Future experience based on work in a large number of laboratories on many more systems will perhaps provide a better guide for selection of appropriate experimental protocols. 5. SUMMARY

In the present article a method is described which allows the delineation of the projections of a single neuron as well as the identification of one or more of its chemical components. The technique is a combination of retrograde tracing and fluorescent dyes based on the work of Kuypers and collaborators and

123 indirect immunofluorescence nally described

histochemistry

by Coons and collaborators.

as origi-

and the National

The cru-

L.S. was a Fogarty-SMRC

Institutes

of Mental Health, postdoctoral

U.S.A.

fellow. We

cial parameters including the selection of the dyes, the injection technique and tissue processing as well

also thank the SMRC for travel funding for L.S. Part

as the appropriate

ty International acknowledged.

Scholar. The scholarship is gratefully The support of Dr. P. Condliffe, Na-

method of choice involves the use of the retrogradely

tional Institutes

of Health,

transported

gratefully

markers

Iodide,

immunohistochemical

and filter combinations

fluorescent

are discussed.

The

dyes Fast Blue, True Blue or Propidium

and in addition,

for double

labeling

ments, Diamidino Yellow or Primuline. combind with FITC (Propidium Iodide)

experi-

They are or TRITC

(Fast Blue, True Blue, Diamidino Yellow, line) as immunofluorescence markers.

Primu-

ACKNOWLEDGEMENTS

The present study was supported by the Swedish Medical Research Council (04X-2887)) Magnus Bergvalls Stiftelse, Knut och Alice Wallenbergs Stiftelse, NINCDS Grant 06801, NIMH Grant ~2714

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