Immunocytochemical detection of two nuclear proteins within the same neuron using light microscopy

Brain Research Protocols 5 Ž2000. 39–48 www.elsevier.comrlocaterbres

Protocol

Immunocytochemical detection of two nuclear proteins within the same neuron using light microscopy Andrew C. McInvale

a, )

, Richard E. Harlan

a,b

, Meredith M. Garcia

a,c

a

Neuroscience Program, Tulane UniÕersity School of Medicine, 1430 Tulane AÕe. SL-2, New Orleans, LA 70112, USA Department of Anatomy, Tulane UniÕersity School of Medicine, 1430 Tulane AÕe. SL-49, New Orleans, LA 70112, USA Department of Otolaryngology, Tulane UniÕersity School of Medicine, 1430 Tulane AÕe. SL-59, New Orleans, LA 70112, USA b

c

Accepted 2 September 1999

Abstract We developed a method of double immunocytochemistry ŽICC. that can be used with conventional light microscopy for localizing two different nuclear proteins. The procedure involves two sequential rounds of ICC that both employ the avidin and biotin conjugated enzyme ŽABC. amplification method, separated by an Avidin D and biotin blocking step to reduce non-specific avidin–biotin reactions. Round one of ICC employs the use of avidin and biotin conjugated alkaline phosphatase ŽABC–AP. and the Vector Red ŽVR. substrate, which produces a red colorimetric reaction product. The second round of ICC makes use of avidin and biotin conjugated peroxidase ŽABC–HRP. and the Vector w SG substrate, which produces a gray colorimetric reaction product. Neuronal nuclei that are double-labeled for both proteins appear red with a gray core. This protocol allows the simultaneous detection of two proteins within the same subcellular compartment of a single neuron, without the need for epifluorescence or scanning confocal laser microscopy. q 2000 Elsevier Science B.V. All rights reserved. Theme: Cellular and Molecular Biology Term: Staining, tracing, and imaging techniques Keywords: Immunocytochemistry; Avidin–biotin complex; Peroxidase; Alkaline phosphatase

1. Type of research Ø Many studies have used immunocytochemistry ŽICC. with enzymatically activated chromagenic substrates to localize two different antigens in separate subcellular compartments w7,10,11,13–17,21,26,30x. Ø A growing number of studies are utilizing immunofluorescence techniques along with either epifluorescence microscopy w2,5,6,8,22,28x or with scanning confocal laser microscopy w1,9,20,25,27,29x in order to localize two different antigens within the same subcellular compartment of a neuron. Ø This protocol was designed to permit the colocalization of two neuronal antigens within the same subcellular compartment, using primary antibodies from different species, avidin–biotin conjugated ŽABC. immunocyto-

chemical procedures, and two different enzyme–substrate reactions walkaline phosphatase ŽAP.rVector Red ŽVR. and peroxidaserVector SGx that produce stable chromogen reaction products. The precipitated chromagens are light, moisture and pH stable, and do not fade with storage. They are easily distinguished by their colors ŽVR is red and SG is gray. and the nature of their precipitate, with VR more diffuse and SG more compact; the double-labeled nuclei thus appear red with a gray core. The procedure requires only standard light microscopy to visualize the antigens. As neither epifluorescence nor confocal laser microscopy is required for visualizing reaction products in this procedure, it is a more generally applicable method for colocalizing antigens in a single subcellular compartment. 2. Time required

)

Corresponding author. Fax: [email protected]

q1-504-582-7846;

e-m ail:

Treating animals and preparing the brain tissue for double ICC takes a variable amount of time. If the experi-

1385-299Xr00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 5 - 2 9 9 X Ž 9 9 . 0 0 0 5 0 - 1

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mental animals need to be subjected to drug or other treatment paradigms, these procedures should be implemented before anesthetizing and perfusing the animal. Each perfusion takes approximately 15 min. Removing the brain takes approximately 5 min. Each brain is postfixed for 2 h, and cryoprotected for approximately 2 days. The double ICC procedure requires 5 days. The first day involves sectioning on a freezing microtome, washing the sections with phosphate-buffered saline ŽPBS., blocking sections with normal donkey serum ŽNDS., and incubating the sections in the first primary antibody. This usually takes approximately 3–5 h. The sections will then incubate for approximately 48 h, and therefore, no procedures are carried out on the second day. Several procedures are carried out on the third day. The sections need to be washed extensively in PBS, incubated in a secondary antibody solution, washed again in PBS, incubated in an avidin and biotin conjugated alkaline phosphatase solution ŽABC–AP., washed in 100 mM Tris pH 8.0 buffer, and then incubated in the VR substrate solution. After these steps are done, the sections need to be washed with PBS, blocked with a solution of Avidin D, rinsed briefly with PBS, blocked with a biotin solution, and then incubated in the primary antibody directed against the second protein of interest. The procedures on day 3 can take approximately 6–8 h. The sections will incubate in primary antibody solution for another 48 h, and no procedures will be done on day 4. Day 5 involves washing the sections, incubating them in secondary antibody solution, washing them in PBS, incubating them in avidin and biotin conjugated peroxidase ŽABC–HRP. solution, washing the sections in PBS pH 7.5, and incubating the sections in Vector w SG substrate. These steps take approximately 4–6 h. Having completed the peroxidase enzymatic reaction, the sections can be stored in PBS until they are mounted onto slides out of Dreft’s detergent solution. This process takes a variable amount of time, depending on the amount of tissue being processed and the thickness of the sections. 3. Materials 3.1. Animals Ø Ø Ø Ø Ø Ø

Breed: CD Sprague–Dawley rats; Breeder: Charles River ŽCambridge, MA.; Sex: male; Age: adult; Weight: 175–200 g; Maintenance: 12L:12D; 228C; free access to food and water.

Ø Ø Ø Ø Ø

Orbital shaker kept at room temperature; Orbital shaker refrigerated at 48C; Microfuge ŽQualitron.; Serological pipettes; Rainin pipettors ŽP10, P200, P1000..

3.3. Chemicals and reagents 3.3.1. Immunological reagents Ø Goat anti-Fos serum ŽSC052G; Santa Cruz Biotechnology.; Ø Rabbit Anti-Androgen Receptor ŽAR. serum ŽPA1111A; Affinity Bioreagents.; Ø NDS ŽJackson ImmunoResearch.; Ø Biotin–SP-conjugated AffiniPure Donkey Anti-Goat IgG ŽH q L. ŽJackson ImmunoResearch.; Ø Vectastain w ABC AP Standard Kit ŽAK-5000; Vector Laboratories.; Ø Vectastain w ABC Elite Rabbit IgG Kit ŽPK-6101; Vector Laboratories.; Ø AvidinrBiotin Blocking Kit ŽSP-2001; Vector Laboratories.; Ø AP Substrate Kit I ŽSK-5100; Vector Laboratories.; Ø Vector w SG Peroxidase Substrate Kit ŽSK-4700; Vector Laboratories.. 4. Detailed procedure 4.1. Animal treatments All experimental procedures involving animals were approved by the Advisory Committee on Animal Resources of Tulane University Medical School and also conformed to NIH guidelines for use of experimental animals. Adult male Sprague–Dawley rats ŽCD strain; Charles River; 175–200 g. were housed in the Tulane Medical School vivarium under regulated light Ž12 h light:12 h dark. and temperature Ž228C. conditions. Animals were given free access to food and water. 1. Deeply anesthetize the rat by injecting it with sodium pentobarbital Ž100 mgrkg i.p... Ensure that the animal is thoroughly anesthetized by pinching the hindlimb with forceps. 2. Transcardially perfuse the animal with PBS pH 7.2 followed by 3% neutral-buffered paraformaldehyde ŽNBPF. as described previously w3x. 3. Remove the brain, postfix in NBPF for 2 h, and cryoprotect in 30% sucrose until the brain sinks. 4. Flash-freeze the brain with crushed dry ice and store at y708C until the double ICC procedure will be performed.

3.2. Special equipment

4.2. Double ICC: round 1, day 1 — cutting sections and adding the first primary antibody

Ø Freezing microtome ŽZeissrMicrom HM 400; Heidelberg, Germany.; Ø Multiple well tissue culture plates ŽCorning.;

1. Cut 40–60 mm serial sections using a freezing microtome into a Corning multiple well tissue culture plate containing PBS.

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2. Wash the sections with PBS 5 = 10 min on an orbital shaker. 3. Dilute the NDS in 0.4% Triton X-100 PBS ŽTX–PBS. to a final concentration of 30 ml NDSrml needed. For 24-well plates, use 0.6 ml per well. For 12-well plates, use a final volume of 1.0 ml per well; and for six-well plates, use a final volume of 2.0 ml per well. 4. Aspirate the PBS, add the NDS, and allow the sections to incubate in the NDS for 1 h at room temperature ŽRT. on an orbital shaker. 5. Dilute the primary antibody that will be used during the first round of ICC with 0.4% TX–PBS. 6. Aspirate the NDS, add the primary antibody solution, and allow the sections to incubate in the primary antibody for 2 days at 48C on an orbital shaker. 4.3. Double ICC: round 1, day 3 and round 2, day 1 — the AP r VR reaction, AÕidin D and biotin blocking, and addition of the second primary antibody Ž1. Aspirate the primary antibody, and wash the sections with PBS 6 = 6 min on an orbital shaker at RT. Ž2. Prepare the secondary antibody solution. If the primary antibody was a polyclonal goat antiserum, then dilute biotinylated donkey anti-goat IgG ŽJackson ImmunoResearch. with 0.4% TX–PBS by a factor of 1:200. If the primary antibody was polyclonal rabbit antiserum, dilute biotinylated goat anti-rabbit IgG ŽVectastainw ABC Elite kit; Vector Laboratories. in 0.4% TX–PBS to a concentration of 2.5 ml secondary antibody per ml of solution needed. If the primary antibody was a monoclonal mouse antiserum, dilute horse anti-mouse IgG ŽVectastain w ABC Elite kit; Vector Laboratories. to a final concentration of 2.5 ml secondary antibody per ml of solution needed. Use the same volume for each well and same total volume that was used during step 3 of day 1. Ž3. Aspirate the PBS and add the secondary antibody. Incubate the sections in the secondary antibody for 1 h at RT on an orbital shaker. Ž4. Dilute the ABC–AP solution in 0.4% TX–PBS to a final concentration of 10 mlrml needed of avidin Žsolution A. and 10 mlrml needed of biotin Žsolution B.. This solution should be made at least 30 min prior to use. The total volume needed is equivalent to the total volume of the secondary antibody solution. Ž5. Aspirate the secondary antibody and wash the sections with PBS 3 = 10 min at RT on an orbital shaker. Ž6. Aspirate the PBS and add the appropriate volume of ABC–AP to each well of the multi-well plate. Allow the sections to incubate in the ABC–AP for 1 h at RT on an orbital shaker. Ž7. Aspirate the ABC–AP and wash the sections 3 = 5 min with 100 mM Tris pH 8.0 on an orbital shaker. Ž8. Prepare the VRrAP substrate by following the directions provided by the manufacturer. The VR substrate is light sensitive, and the enzymatic reaction runs best

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when the substrate is made up fresh. Therefore, prepare the solution immediately before use in a Falcon tube wrapped in aluminum foil. Ž9. Transfer the sections to either crucibles resting in a dish containing Tris pH 8.0 or to wells of a new multi-well tissue culture plate containing Tris pH 8.0. Ž10. Incubate the sections in either another dish or wells containing the VR substrate. Try to keep this reaction as protected from light as possible Že.g., by placing the lid of a box over the dish or well plate.. Start by incubating the sections for a short period of time Že.g., 0.5–1 min., transferring the sections back to Tris pH 8.0, and observing a section under the microscope to judge signal and background staining intensities. Incubating sections for too long may increase background staining without increasing signal staining intensity, hence shorter incubation times are strongly recommended. Ž11. After completing the APrVR reaction, wash the sections 2 = 10 min in PBS at RT on an orbital shaker. Ž12. Prepare the next primary antibody that will be used for the second round of ICC by diluting it in 0.4% TX–PBS. The same final volume for each well should be used as in day 1 step 3. Ž13. Aspirate the PBS and add a volume of Vector Avidin D solution that is sufficient to cover all sections in each well. Allow the sections to incubate in this Avidin D solution for 15 min at RT on an orbital shaker. Ž14. Briefly wash the sections in PBS. Ž15. Aspirate the PBS and add a volume of the Vector biotin blocking solution sufficient to cover all sections in each well. Allow the sections to incubate in the biotin solution for 15 min at RT on an orbital shaker. Ž16. Aspirate the biotin blocking solution and add the appropriate volume of primary antibody to each well. Allow the sections to incubate in the primary antibody for 2 days at 48C on an orbital shaker. 4.4. Double ICC: round 2, day 3 — the peroxidaser Vector SG reaction Ž1. Remove the primary antibody and wash the sections with PBS 6 = 6 min at RT on an orbital shaker. Ž2. Dilute the secondary antibody. For polyclonal goat primary antibodies, dilute biotinylated donkey anti-goat IgG ŽJackson ImmunoResearch. in 0.4% TX–PBS by a factor of 1:200. For polyclonal rabbit primary antibodies, dilute biotinylated goat anti-rabbit IgG ŽVectastain w ABC Elite kit; Vector Laboratories. in 0.4% TX-PBS to a concentration of 2.5 ml secondary antibody per ml of solution needed. For monoclonal mouse primary antibodies, dilute horse-anti mouse IgG ŽVectastain w ABC Elite kit; Vector Laboratories. to a final concentration of 2.5 ml secondary antibody per milliliter of solution needed. The total volume needed for the secondary antibody solution will be equal to the volume of blocking solution used in day 1 step 3.

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Ž3. Remove the PBS and add the appropriate volume of the secondary antibody solution to each well. Allow the sections to incubate in the secondary antibody for 1 h at RT on an orbital shaker. Ž4. Prepare the ABC–HRP solution. The same ABC solution ŽVectastain w ABC Elite kits; Vector Laboratories. can be used for any secondary antibody solution. Dilute the ABC solution in 0.4% TX–PBS to a concentration of 10 mlrml needed of the avidin solution and 10 mlrml needed of the biotin solution. This solution should be prepared at least 30 min prior to use. The total volume needed is the same as the volume used for the secondary antibody solution. Ž5. After the 1 h incubation period, remove the secondary antibody solution and wash the sections with PBS 3 = 10 min at RT on an orbital shaker. Ž6. Remove the PBS and add the appropriate volume of ABC–HRP solution to each well. Allow the sections to incubate in ABC–HRP solution for 1 h at RT on an orbital shaker. Ž7. Aspirate the ABC solution and wash the sections with PBS pH 7.5 3 = 5 min at RT on an orbital shaker. Ž8. Prepare the Vector w SG substrate ŽVector Laboratories. according to the directions provided by the manufacturer. This substrate is light sensitive and should be prepared in a Falcon tube that is light protected Že.g., wrapped in aluminum foil.. Furthermore, the peroxidase reaction runs best with freshly prepared substrate, therefore make the substrate solution immediately before it is needed. Ž9. Transfer the sections either to crucibles submerged in a dish of PBS pH 7.5 or into wells containing PBS pH 7.5 from a new multi-well tissue culture plate. Ž10. Perform the peroxidaserVector w SG enzymatic reaction. Keep the reaction light protected Že.g., by covering the crucibles or well plate with a box.. Start out by incubating for short reaction times Že.g., 0.5–1 min. and observing signal and background staining under a light microscope. Incubating sections for too long can create too much background staining and therefore should be avoided if possible. Ž11. After the peroxidase reaction has been completed, the sections can be temporarily stored in PBS pH 7.5 at 48C until they can be mounted onto slides. However, the sections should be mounted onto slides as soon as possible, because PBS will evaporate and cause sections to become dehydrated.

Ž12. Transfer the sections into a Dreft’s detergent solution Ž0.1% Dreft’s detergent in 10 mM NaOAc. and mount them onto subbed slides. Dry the slides on a slide warmer, dehydrate the slides in 100% ethanol, clear the slides in Histo-Clear w , and coverslip the slides using Permount w mounting medium. After allowing the slides to dry, the slides can be viewed and photographed with conventional light microscopes. The reaction products of both substrates are stable and do not fade with prolonged storage or exposure to light.

5. Results The type of result obtained from the ICC procedures described in this protocol is the localization of two different nuclear proteins in the brain. The distributions of two proteins can be examined, and possible colocalization of the two proteins within single neurons of a particular brain region can be assessed using a light microscope. 5.1. Single-labeled neurons with AP r VR The APrVR enzymatic reaction produces a red colorimetric reaction product. An example of neurons that are single-labeled with the APrVR method is provided in Fig. 1A. In this example, Fos-like immunoreactivity is detected in a region of the midline intralaminar thalamus from a rat treated with ethanol Ž5 grkg i.p.. w19x. 5.2. Single-labeled neurons with peroxidaser Vector SG The Vector w SG substrate produces a gray reaction product when reacted with ABC peroxidase. An example of neurons single-labeled with this method is illustrated in Fig. 1C. Here AR immunoreactivity is detected in the bed nucleus of the stria terminalis of a rat treated with ethanol Ž5 grkg i.p.. w19x. 5.3. Double-labeled neurons The nuclei of neurons that are double-labeled for two nuclear proteins by using both the APrVR and the peroxidaserVector w SG method appear red with a gray core. Hence, two different proteins can be detected within the same subcellular compartment of a single neuron. Exam-

Fig. 1. Photomicrographs of coronal brain sections taken from the brain of a rat injected with ethanol Ž5 grkg i.p... ŽA. Fos-like immunoreactivity in the midline intralaminar thalamic nuclei detected by the ABC–APrVR method. ŽB. Elimination of specific staining by incubation in Avidin D and biotin solutions prior to ABC–APrVR steps. ŽC. AR immunoreactivity in the bed nucleus of the stria terminalis detected by the ABC–HRPrVector w SG method. ŽD. Elimination of specific immunoreactivity by performing the Avidin D and biotin blocking steps prior to the ABC–HRPrVector w SG steps. ŽE. Colocalization of Fos ŽABC–APrVR. and AR ŽABC–HRPrVector w SG. in a subset of neurons within the arcuate nucleus. Note the presence of single-labeled Fos neurons Žred; open arrow., single-labeled AR neurons Žgray; closed arrow., and double-labeled neurons Žred with gray core; two arrows.. ŽF. Another example of double-labeled neurons located within the sublenticular extended amygdala. Scale bars in panels A–D represent 100 mm; scale bars in E and F represent 50 mm.

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ples of double-labeled neurons are depicted in Fig. 1E and F, where ethanol-induced Fos expression is demonstrated to be colocalized with AR expression within a subset of neurons in the arcuate nucleus and sublenticular extended amygdala ŽSLEA. of a rat given ethanol Ž5 grkg i.p.. w19x. The same results were obtained whether the immunostaining for c-Fos was performed first followed by AR, or immunostaining for AR was performed first followed by c-Fos. In sections where double-labeled neurons were observed, single-labeled neurons for Fos and AR were also observed, indicating that there was no cross reactivity between reagents. 5.4. AÕidin D and biotin blocking control Since both rounds of ICC employ ABC enzymes as immunodetectors, there is the possibility that non-specific avidin–biotin interactions can occur between the biotinylated secondary antibodies and ABC reagents used in the two rounds of ICC. In order to reduce non-specific avidin–biotin reactions, this protocol adopts Avidin D and biotin blocking steps before the second round of ICC begins. In order to test how effective these blocking steps were in removing free avidin and biotin binding sites, some sections were incubated with the primary antibody, then biotinylated secondary antibody, followed by the avidin–biotin blocking step. Sections were then incubated with ABC–AP or ABC–HRP, followed by the appropriate substrate. No immunostaining was observed in these sections, demonstrating that all free biotin molecules on the secondary antibody had been successfully blocked. Fig. 1B illustrates an example of a section incubated in Avidin D and biotin solutions prior to APrVR visualization of Fos immunoreactivity. The blocking steps effectively remove all specific staining for Fos in the section. Similar control experiments were performed on single-labeled sections stained for AR by the peroxidaserVector w SG method. Blocking sections with Avidin D and biotin solutions prior to the ABC–HRP incubation and peroxidaserVector w SG reaction completely eliminated specific staining ŽFig. 1D..

6. Discussion 6.1. Troubleshooting As with all immunocytochemical procedures employing enzyme–substrate reactions, background staining may be a problem. However, the Avidin D and biotin blocking step after the first round of ICC reduces non-specific background staining considerably ŽFig. 1B,D.. Primary antibodies should be diluted as much as possible in order to minimize background. Experimenting with different incubation times during either of the substrate–enzyme reaction steps will also be critical for optimizing the signal to noise ratio. Trying different buffers may also decrease

background staining intensity. For instance, we switched from 100 mM Tris pH 8.2 to 100 mM Tris pH 8.0 and had superior results for the APrVR reaction combination. 6.2. AlternatiÕe and support protocols An alternative to the ABC methods described in this protocol is the peroxidase–antiperoxidase ŽPAP. method w23x. Although detection of antigens by the PAP method can provide experimenters with approximately three-fold amplification of signal and little background, the PAP method relies on a free binding site on the secondary antibody. Thus, if the primary antibody concentration is too high, then the secondary antibody will recognize two primary antibodies Žoften referred to as ‘‘bridging’’. and the PAP complex will not have access to the secondary antibody. Furthermore, we have also found that the sensitivity of this technique may not always be as high as the ABC method, depending on the specific antigens in question. A number of alternative methods exist for detection at the light level of multiple antigens in different subcellular compartments of a single neuron, but relatively few are suitable for detection of more than one antigen in a single subcellular compartment. One approach used is the socalled ‘‘mirror technique’’ w6,12x. In this approach, adjacent sections were immunostained for different antigens. Cells bisected in the plane of section were identified in the paired surfaces of the adjacent sections and identified as containing immunoreactivity for each antigen in the different halves of the cell. This was accomplished by preparing photomontages of the sections. Sections containing neurons with perikarya on the cut surface of each section were then examined under 100 = oil immersion and the perikarya marked on the photomontage according to whether the cells were essentially bisected Ž‘‘group 1’’ cells., or had the majority of the cell contained in that section Ž‘‘group 2’’ cells. or the adjacent section Ž‘‘group 3’’ cells.. Only ‘‘group 1’’ cells were subjected to further analysis, by doing oil immersion camera lucida drawings of the cells and any nearby landmarks such as blood vessels. By registration of perikarya and other landmarks on camera lucida drawings of adjacent sections, it was possible to determine which neurons expressed both types of immunoreactivity. The validity of the method was established by performing the analysis on adjacent sections immunostained with a single antibody. A more widely applicable approach to labeling multiple antigens in a single tissue section involves the use of different chromagens. Lanciego and Giminez-Amaya w16x describe a double-labeling procedure for colocalizing the retrograde tracers cholera toxin B subunit ŽCTB. or fluoro-gold ŽFG. with tyrosine hydroxylase ŽTH. using the PAP procedure, with DAB Ždiaminobenzidine; brown reaction product. as the chromagen for detection of CTB or FG and Vector-VIP Žpurple reaction product. as the chroma-

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gen for TH. Using this procedure, they were able to demonstrate CTB in soma and proximal dendrites of neurons that also showed TH-IR in distal dendrites. They noted that the order in which the substrates are used is critical, with DAB masking the VIP reaction product when DAB staining is done first; this is in contrast to our method, where we saw no differences in staining whether the order was VR followed by SG, or SG followed by VR. Because Lanciego and Giminez-Amaya used two substrates that are peroxidase-based, they also reported a color-mixing phenomenon, in which there is bleed-through of the two chromagens. As our method uses one AP substrate and one peroxidase substrate, color-mixing or bleed-through does not occur, as each substrate is precipitated by a different enzyme. Zhou and Grofova w30x also used two peroxidase substrates, DAB and Vector-VIP or SG to co-localize the anterograde tracer, PHA-L, the retrograde tracer, CTB, and choline acetyltransferase ŽChAT. in individual neurons. They found that while VIP and DAB were suitable for co-detection of antigens in different neuronal compartments, VIP and DAB could not be distinguished in the same subcellular compartment; this is in contrast to our method, where nuclei labeled with both VR and SG can be distinguished from nuclei labeled with only one substrate. They also reported that the order of substrate was critical, with DAB staining performed before VIP staining. Lanciego et al. w15x combined VIP with DAB or nickel-intensified DAB ŽNi-DAB; black reaction product. to immunodetect the retrograde tracers biotinylated dextran amine ŽBDA. or FG with the calcium binding proteins, parvalbumin or calbindin D28k. Ni-DAB was used for detection of BDA, followed by PAP–DAB for FG, and ABC–peroxidase ŽABC–P. –VIP for the calcium binding proteins. They also reported that the order in which the chromagens were used was critical, and that the VIP reaction product is lost during ethanol dehydration prior to clearing and coverslipping. In our procedure, neither VR nor SG was found to be affected by ethanol dehydration. Double-immunostaining with substrates other than DAB, VIP and SG have also been used, but with less successful results. In a heroic study, Trojanowksi et al. w26x compared eight different peroxidase substrate protocols in ICC, using the PAP method, concluding that DAB with imidazole enhancement provided the best sensitivity for ICC, while tetramethylbenzidine ŽTMB; blue reaction product. was superior for visualization of horseradish peroxidase used as a tract tracer. Norgren and Lehman w21x combined the peroxidase substrates TMB and DAB to identify neuropeptide Y terminals on LH–RH neurons in the hypothalamus, but found TMB to be very unstable at pH greater than 3.3; this could be somewhat overcome by coupling the TMB to molybdic acid, but even this required that the peroxidase reaction be carried out at pH 6 or below. The TMB reaction product is also unstable with storage, and faded within days when sections were coverslipped in glycerol. We also attempted to use TMB in developing our double-

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label procedure, and experienced similar problems with instability of the reaction product and fading. Levey et al. w17x used the PAP method, combining DAB and benzidine dihydrochloride ŽBDHC. to localize ChAT and TH or substance P ŽSP.. BDHC is also a less stable substrate than DAB, VIP or SG. The color of the BDHC reaction product varies with the pH: at pH - 7.0 it is blue, while at pH ) 7.0, it is brown and resembles DAB; even after the development step, if it is exposed to a pH above 7.0, it will turn brown. It is also sensitive to the type Žphosphate vs. acetate. and molarity of the buffer used, and gives higher background staining than DAB. Furthermore, it nonspecifically stains nuclei w14,17x, which makes it unsuitable for immunostaining of nuclear antigens such as c-Fos. Levey et al. w17x also noted that the intensity of the DAB and BDHC deposits would make it difficult to resolve both chromagens in the same structure, in contrast with our method. Lakos and Basbaum w14x also combined DAB and BDHC as chromagens for immunodetection of different antigens in the pre- and post-synaptic elements of dendrites, finding that this combination was particularly useful at the electron microscopic ŽEM. level, as the quality of the precipitates formed Žcrystalline BDHC vs. flocculent DAB. make them easy to distinguish with the EM. They noted that the chemistry of the BDHC reaction requires that the DAB reaction be carried out first, although the intensity of the BDHC reaction product may mask the DAB in double-labeled neurons, making it less suitable for immunodetection of two antigens in the same neuronal compartment. Hancock w7x used sequential rounds of PAP immunostaining with Ni-DAB followed by DAB to look at codistribution Žvs. colocalization. of antigens in the same tissue section, finding that the Ni-DAB step had to be performed first to prevent it from obscuring the DAB reaction product. Fewer studies have combined peroxidase substrates with substrates activated by other enzymes for labeling of more than one antigen. However, in a beautiful series of experiments, Joseph and Piekut w11x combined PAP–DAB with glucose oxidaseranti-glucose oxidase ŽGAG.rnitroblue tetrazolium ŽNBT; blue reaction product. to examine the codistribution of a number of antigens ŽACTH, CRF, TH, 5-HT, oxytocin, and AVP. in the mesencephalon. While they obtained superior results with this method, it should be noted that they did not attempt colocalization of these antigens in a single neuron, as we were able to achieve with our method. Also, they noted that the sensitivity of the PAP method was not as good as the ABC method, possibly because the size of the PAP or GAG complex was too large to permit good tissue penetration. We also found that in our system, using PAP resulted in reduced sensitivity, which is the reason that we chose to use two ABC coupled enzymes with an avidin–biotin blocking step intervening. Another possible method that could be used to detect two antigens in the same subcellular compartment would

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combine peroxidase–chromagen ICC with radioimmunocytochemistry ŽRICC.. RICC has been described using second antibodies labeled with either 3 H w4x or 35 S w18x, followed by emulsion autoradiography. While the cellular resolution of the signal obtained with 3 H is superior to that obtained with 35 S, the longer exposure times required for 3 H autoradiography, in addition to the problem of disposal of 3 H radioactive waste, make it less attractive than 35 S. The degree of resolution of radioimmunoreactivity detected with 35 S at the subcellular level would determine how useful this method would be when combined with standard methods of ICC in detecting colocalization of antigens in the same subcellular compartment. Immunodetection of more than one antigen using ICC with fluorescent antibodies has been extensively used, with microscopes equipped with epifluorescence as well as confocal laser scanning microscopes. Typically, the procedures using epifluorescence utilize second antibodies conjugated to different fluorophores, and immunodetection is accomplished using photomicroscopy of the tissue with different sets of excitationremission filters. Campbell et al. w2x used second antibodies coupled to rhodamine and fluorescein isothiocyanate ŽFITC. combined with the True Blue retrograde tracer to visualize TH, GAD and True Blue labeled neurons, taking photomicrographs of the same section with three different sets of filters. In a similar study, Hajos et al. w6x colocalized the M2 subtype of the muscarinic acetylcholine receptor ŽAChRM2. with parvalbumin, calbindin D28k, VIP, CCK, GABA, or calretinin, using a FITC-coupled second antibody for immunodetection of AChRM2 and a rhodamine-coupled second antibody for immunodetection of the other antigens. While this approach was successful, it should be noted that AChRM2 and the other antigens were present in different subcellular compartments. It should be noted that there is no signal amplification step used in the above procedures, so the use of fluorescent second antibodies to immunodetection may be limited to those antigens that are present at more abundant levels. Photobleaching — loss of signal upon exposure to light — is also a problem with many fluorophores, more so with FITC and AMCA Žblue fluorophore. than with more recently developed compounds such as Cy2 Žgreen fluorophore. and Cy3 Žred fluorophore., as reported by Hartig et al. w8x. In this elegant study, an amplification step was also possible, as the primary antibodies were first conjugated to biotin or digoxigenin, permitting the use of strepavidinrCy3 or anti-digoxigeninrFITC. Immunodetection was also performed using standard fluorophore-labeled second antibodies Že.g., goat anti-rabbit IgG coupled to AMCA., permitting triplelabel immunofluorescent ICC in the same section. A similar method was used for double-label fluorescent ICC by Wurden and Homberg w28x. Two potential problems exist with the approach of labeling the primary antibodies to improve sensitivity of fluorescence ICC. One relates to the amount of antibody that is available. For commercially

available antibodies, this is of only economic concern, but for antibodies that are provided as gifts by collaborators, the supply may be too limited to permit this approach. The second potential problem relates to the purity of the antibody — if the purified IgG fraction is unavailable, then this approach may not be possible. The use of fluorescent labels for immunodetection of multiple antigens in a single neuron has been greatly facilitated by the advent of confocal laser scanning microscopy ŽCLSM.. Confocal microscopy prevents false positives in immunodetection of double-labeled cells that can occur in conventional fluorescence microscopy when single-labeled cells are superimposed on one another in the Z-plane but appear to be double-labeled because the depth of focus is low in conventional microscopy w27x. The brief exposure to the laser beam that occurs during the scan Ž100 scans in 2.7 ms vs. up to 30 s exposure with conventional epifluorescence microscopy. also permits the successful use of even light-sensitive compounds such as FITC in fluorescent ICC w1,20,24x. In a study by Mossberg et al. w20x, 5-HT, SP, enkephalin, and TRH were localized or colocalized in axon terminals of the cat spinal cord, using second antibodies conjugated to FITC or TRITC Ža red fluorophore.. As the spectra of FITC and TRITC overlap somewhat, the laser power and wavelengths used were optimized such that both FITC and TRITC could be used without ‘‘shine-through’’ of the fluorescent signals. While this reduced the FITC signal somewhat, requiring multiple scans to acquire a signal of sufficient strength, the short exposure time to the laser did not cause significant photobleaching. Nevertheless, with double-label fluorescence ICC requiring two series of scans at multiple levels of the tissue, the authors recommended restricting the number of optical layers analyzed to reduce photobleaching. Combining three fluorophores chosen for maximum separation of spectra ŽCy5, fluorescein and rhodamine., Bausch et al. w1x were able to successfully colocalize parvalbumin, mu opioid receptor, and the lectin Vicia Õillosa agglutinin in single neurons of rat brain. Wouterlood et al. w27x also reported successful and unequivocal colocalization of calretinin and GABA in single neurons using Cy2 and Cy5, fluorophores with non-overlapping absorptionremission spectra and two-laser CLSM. Compared to a single-laser approach using Cy2 and Texas Red, the Cy2rCy5 procedure was found to be superior, as there was no ‘‘shine-through’’ with the latter fluorophores, while this did occur with Cy2rTexas Red. Finally, in a study by Todd w24x, CLSM immunofluorescence ICC was combined with peroxidaserDAB ICC to permit ultrastructural analysis of multiple antigens in the same cell. Here, triple-label CLSM ICC was performed using primary antibodies raised in three species Žrabbit anti-NK1 receptor, sheep anti-CGRP, and rat anti-SP., detected with second antibodies labeled with FITC, rhodamine, and Cy5. Following CLSM analysis, the tissue sections were reacted with biotinylated second antibodies and streptavidin–

A.C. McInÕale et al.r Brain Research Protocols 5 (2000) 39–48

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peroxidase, followed by DAB. Low magnification EM photographs were taken to match DAB immunoreactivity with that visualized with the confocal microscope, followed by higher magnification EM analysis to determine ultrastructural localization. This permitted ultrastructural analysis of more than one antigen even though the antigens were all immunodetected with the same chromagen. While the use of CLSM unquestionably provides superior resolution of multiple antigens in a single cell, this technology is not universally available. Our method provides an alternative that can be used with the standard light microscope, an item of equipment present in every neuroanatomy laboratory.

3. Add secondary antibody solution, incubate sections for 1 h. 4. Prepare ABC–HRP solution. 5. PBS washes: 3 = 10 min. 6. Add ABC–HRP solution, incubate sections for 1 h. 7. PBS pH 7.5 washes: 3 = 5 min. 8. Prepare the Vector w SG substrate. 9. Transfer sections to crucibles or a multiple well plate containing PBS pH 7.5. 10. Peroxidase–Vector w SG reaction. 11. Store sections in PBS pH 7.5 at 48C until mounting onto slides. 12. Mount sections onto subbed slides.

7. Quick procedure

8. Essential references

7.1. Double ICC: round 1, day 1 — cutting sections and adding the first primary antibody 1. 2. 3. 4. 5. 6.

Cut serial brain sections on a freezing microtome. PBS washes: 5 = 10 min. Prepare NDS. Add NDS, incubate sections for 1 h. Dilute primary antibody. Add the primary antibody, incubate the sections for 2 days at 48C.

7.2. Double ICC: round 1, day 3 and round 2, day 1 — the AP r VR reaction, AÕidin D and biotin blocking steps, and addition of the second primary antibody 1. PBS washes: 6 = 6 min. 2. Prepare secondary antibody solution. 3. Add the secondary antibody, incubate the sections for 1 h. 4. Prepare ABC–AP solution. 5. PBS washes: 3 = 10 min. 6. Add the ABC–AP solution, incubate the sections for 1 h. 7. Tris pH 8.0 washes: 3 = 5 min. 8. Prepare the VR substrate. 9. Transfer sections to Tris pH 8.0 in crucibles or a new multi-well plate. 10. APrVR reaction. 11. PBS washes: 2 = 10 min. 12. Prepare the second primary antibody. 13. Avidin D block for 15 min. 14. Quick PBS wash. 15. Biotin block for 15 min. 16. Add the second primary antibody, incubate sections for 2 days at 48C. 7.3. Double ICC: round 2, day 3 — peroxidase–Vector w SG reaction 1. PBS washes: 6 = 6 min. 2. Prepare secondary antibody solution.

Refs. w3,23,29x.

Acknowledgements This work was supported by LEQSF RD-A-29 awarded to M.M.G.

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