A non-radioactive in situ hybridization method that does not require RNAse-free conditions

Journal of Neuroscience Methods 85 (1998) 129 – 139

A non-radioactive in situ hybridization method that does not require RNAse-free conditions Enrico Tongiorgi a,b,*, Massimo Righi a, Antonino Cattaneo a a

International School for Ad6anced Studies (SISSA), Neuroscience Program, Via Beirut 2 /4, 34014 Trieste, Italy b Department of Biology, Uni6ersity of Trieste, Via Giorgieri 10, 34127 Trieste, Italy Received 12 March 1998; received in revised form 13 July 1998; accepted 19 July 1998

Abstract This report describes a quick and versatile method to perform non-radioactive in situ hybridization in which none of the hybridization steps are performed under RNAse-free conditions. This study demonstrates that in situ hybridization can be performed without an RNAse-free environment provided that the concentration of RNAse introduced during the experiment does not reach 0.1 mg/ml, a concentration that is unlikely to be achieved through an accidental contamination. Moreover, evidence is provided that the only step sensitive to RNAse degradation is the pretreatment since degradation during the hybridization step can not occur due to a very efficient protective effect exerted by formamide. Finally, our data suggest that endogenous RNAse activity might be readily neutralized through paraformaldehyde fixation. A feature of this method is the strong fixation that ensures a perfect tissue preservation, even at level of the fine structure of the cell processes. The method allows a uniform tissue penetration by sodium periodate and sodium borohydride treatment and can be easily used in combination with diaminobenzidine immunohistochemistry for double labeling experiments. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Digoxigenin; Biotin; TrkA; TrkB; b-Actin; Myelin basic protein; Dendritic mRNA; Rat basal forebrain; Rat hippocampus

1. Introduction In situ hybridization is a powerful technique that enables the visualization of a nucleic acid sequence (either DNA or RNA) in target tissues, cells, nuclei, organelles or chromosomes by means of a complementary nucleic acid probe. Initially, this technique was developed to detect ribosomal DNA sequences in the nuclei of cultured cells, using a radiolabeled ribosomal RNA probe (Gall and Pardue, 1969; John et al., 1969). The first study describing the detection of an mRNA by in situ hybridization was that by Harrison et al. (1974) who used a tritiated cDNA-probe to detect the globin mRNA in mouse fetal liver cells. Ten years later, Cox et al. (1984) performed * Corresponding [email protected]

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in situ hybridization with radioactive single stranded cRNA probes (riboprobes) to detect histone mRNAs in sea urchin embryos. Later, non-radioactive in situ hybridization methods have been developed, first using biotin-labeled (Albertson, 1985), and then digoxigeninlabeled probes (Tautz and Pfeifle, 1989). These simple and poorly dangerous methods have received increasing consensus since the recent demonstration that they are equally sensitive as the radioactive ones (Bartsch et al., 1992; Emson, 1993; Karr et al., 1995). Moreover, the non-radioactive methods appear particularly useful in those studies aiming at determining the subcellular localization of mRNAs since they provide a much higher spatial resolution of label in less time than that obtained with radioactive methods (Ainger et al., 1993; Emson, 1993; Bian et al., 1996). Despite the recent description of simplified radioactive and non-radioactive protocols (Eberwine et al.,

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1994), in situ hybridization is still considered a laboratory intensive and challenging technique. Among the various difficulties of this technique, the possibility that the probe or the target mRNA might be degraded by a residual endogenous RNAse activity or by accidental RNAse contamination, is considered one of the major potential causes of experimental failure (Blumberg, 1987). Thus, to preserve the target mRNA or the riboprobe from the risk of RNAse degradation, generally all in situ hybridization protocols emphasize that the whole procedure must be performed under RNAsefree conditions. This requirement has the consequence to sensibly increase the cost and the complexity of the methods since it obliges the experimenter to frequently change the disposable material, to use a separate set of RNAse-free treated laboratory tools, and RNAse-free solutions. In order to simplify this technique, this report describes a simple and highly sensitive method that can be performed without using RNAse-free conditions. Furthermore, in this study the real risk of loss of signal due to RNAse degradation is assessed on both target RNA and riboprobe by performing various steps of this in situ hybridization protocol in the presence of RNAse.

2. Materials and methods

2.1. Riboprobes The 700 bp rat b-actin clone (Nudel et al., 1983) was provided by Dr R. Possenti (Institute of Neurobiology CNR, Rome, Italy). The mouse TrkA clone pDM97 (Holtzman et al., 1992) was 480 nucleotides long, and was provided by Dr C.K. Chen (John Hopkins University Medical School, Baltimore, MD). The rat TrkB cDNA clone was provided by Dr Y. Bozzi (Institute of Neurophysiology, CNR, Pisa) (Bozzi et al., 1995), and contained the first 238 bp of the region coding for the tyrosine–kinase domain (nucleotides 2163 – 2401, Middlemas et al., 1991). The plasmid containing the insert coding for the rat myelin basic protein was provided by Dr L. Wrabetz (DIBIT-San Raffaele, Milan, Italy). After linearization of 5 mg the plasmids with the appropriate restriction enzyme in a 30-ml restriction mixture, the volume was increased to 200 ml with H20 and then the solution was extracted with an equivalent volume of phenol, followed by an extraction with chloroform/phenol (1:1 v/v) and chloroform alone. Plasmids were ethanol precipitated at −20°C for 2 h, centrifuged, speed-vac dried 15 min and dissolved in 10 ml of diethylpirocarbonate-treated (DEPC) ultra pure H2O. These templates were stored at −20°C and in our experience remained unaltered for years. The digoxigenin labeled riboprobes were synthesized with a SP6/ T7 DIG-RNA labeling kit (Boehringer) according to

the manufacturer’s instructions. The biotin labeled riboprobes were synthesized as the digoxigenin labeled ones by using the biotin–NTP mix (Boehringer) instead of the digoxigenin–NTP mix purchased with the SP6/ T7 DIG-RNA labeling kit. We found that higher yields of riboprobe (approximately 10 mg) can be reached when the riboprobes are synthesized under RNAse-free conditions and we recommend to store them dissolved in autoclaved ultra pure DEPC–H2O with added RNAsin, at − 20°C.

2.2. Perfusion and tissue processing Adult Wistar rats were deeply anesthetized with 10.5% chloral hydrate (Merck) in physiological solution at a dose of 0.1 ml/100 g body weight and perfused trough the ascending aorta first with a physiological solution followed by 4% paraformaldehyde (PFA, Merck) in phosphate buffer saline (PBS) pH 7.4. After perfusion the brains were removed from the cranial cavity, postfixed and cryoprotected at least overnight at 4°C in a solution of 4% PFA/20% sucrose in PBS. Brains were stored at 4°C in this solution up to 6 months. Brains were quickly frozen with CO2, and serial 40-mm thick slices were cut at the cryomicrotome Histoslide 2000R (Leica). Free-floating sections were processed for in situ hybridization starting with a postfixation for 3 h in 4% PFA in PBS at room temperature (RT) or were stored up to 12 months in a solution of 4% PFA in PBS at 4°C.

2.3. Definition of RNAse-free and non-RNAse-free conditions For the in situ hybridization experiments under RNAse-free (RF) conditions all the solutions were prepared with ultrapure water (upH2O, defined as below) treated with 0.1% diethylpyrocarbonate for 6 h (DEPC–upH2O) and then autoclaved for 1 h at 120°C. Glassware and pipette plastic tips were autoclaved 1 h at 120°C and only sterile, RF disposable plasticware was used. Disposable plasticware was used once and then discarded. Gloves were worn during all steps and were continuously changed. Solutions were made with RF compounds used only for in situ hybridization purposes, dissolved in DEPC–upH2O and when possible autoclaved 1 h at 120°C. The freezing microtome blade and the teflon/plexiglas baskets (plexiglas cylinders sealed with a fine Teflon mesh at the bottom) used to hold the free floating sections were incubated 1 h with 0.1 M NaOH and accurately rinsed with DEPC– upH2O prior the use. Sections were transferred with sterile glass hooks or thin brushes rinsed once with 0.1 M NaOH followed by rinsing several times with DEPC–upH2O.

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For the in situ hybridization experiments carried out under non-RF conditions, non-autoclaved upH2O was used instead of DEPC – upH2O. UpH2O is type I reagent grade water having resistivity of 18.2 MV/cm and total organic carbon (TOC) less than 30 ppb. In our case the upH2O was obtained from tap water pretreated first by reverse osmosis (RO) with a MilliRO-10 Plus (Millipore) purification device and subsequently with a Milli-Q Plus (Millipore) purification device. Solutions were not DEPC treated and were autoclaved maximally for 20 min at 120°C, just to prevent the development of contaminant bacteria or molds. Glassware and plasticware were not autoclaved with the exception of plastic pipette tips which were sterilized by autoclaving for 20 min at 120°C. Sections were transferred with thin brushes or non-sterile glass hooks that were reused several times. Gloves were used only during the handling of hazardous solutions such as paraformaldehyde, sodium periodate and sodium borohydride. Plasticware and teflon/plexiglas baskets for free-floating handling of sections were reused several times without any special treatment provide they were washed as follows. First, labware was washed with tap water and a neutral cleaning agent. Different cleaning agents such as Ausilab101 (Carlo Erba) or RBS-neutral (Roth) were routinely used without noticing any difference in the in situ staining quality. After this first wash the labware was briefly rinsed in deionized water obtained with a MilliRO-10 plus device and finally was thoroughly rinsed with upH2O. The freezing microtome blade was briefly cleaned with a non-sterile paper towel wetted with commercial denatured alcohol.

2.4. Pretreatment of slices under RNAse free or non-RNAse free conditions Prior to the hybridization, slices underwent a permeabilization procedure carried out either in 6- or 12-wells plastic tissue culture plates (Costar) or in teflon/plexiglas baskets immersed in glass beakers. Free-floating slices were washed twice in 0.1% Tween 20 in 1 × PBS (PBST) at RT for 5 min, and quickly washed in H2O, then the slices were permeabilized with 2.3% sodium meta-periodate (Sigma) in H2O at RT for 5 min and quickly washed in H2O. After these steps the sections were incubated in 1% sodium borohydride (Sigma) in 0.1 M Tris–HCl buffer pH 7.5 at RT for 10 min, washed twice in PBST for 3 min at RT. Then the slices were digested with 8 mg/ml proteinase K (BoehringerMannheim) in PBST at RT for 10 – 20 min (exact times should be empirically determined with each proteinase K batch) and washed twice in PBST at RT for 5 min. After digestion the tissue sections were fixed in 4% PFA in PBS at RT for 5 min and washed three times in PBST at RT for 10 min.

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2.5. Hybridization Prehybridization was carried out at 55°C for 60–90 min in plastic multiwell plates (12-wells, Costar) in the prehybridization solution, containing: 20 mM Tris– HCl (pH 7.5), 1 mM EDTA (Sigma), 1 × Denhardt’s solution, 300 mM NaCl, 100 mM dithiothreitol (Sigma), 0.5 mg/ml Salmon sperm DNA (Sigma), 0.5 mg/ml polyadenylic acid (Sigma) and 50% formamide (Sigma). Slices were then transferred with glass hooks into the hybridization solution, composed by the prehybridization solution additioned with 10% dextransulphate, and 50–100 ng/ml digoxigenin or biotin labeled riboprobes. The riboprobes were always pipetted into the hybridization solution with RNAse-free pipette-tips to avoid the risk of RNAse degradation of the probes. In situ hybridization was carried overnight (at least 16 h) in multiwell plates at 55°C without agitation.

2.6. Post hybridization and detection After hybridization the sections were washed twice in 2× saline sodium citrate, 0.1% Tween 20 (SSCT)/50% deionized formamide at 55°C for 30 min, 20 min in 2× SSCT at 55°C and twice in 0.2 SSCT at 60°C for 30 min. Slices hybridized with digoxigenin labeled riboprobes were processed for immunodetection with an anti-DIG antibody F(ab)2 fragment conjugated with alkaline phosphatase, (Boehringer), diluted 1:500 in PBST containing 10% fetal calf serum (FCS) at 4°C overnight. After this incubation the sections were washed four times in PBST at RT for 10 min, then incubated in developing buffer (0.1 M Tris–HCl buffer, 0.1 M NaCl, 0.05 M MgCl2, 1 mM Levamisol) at RT for 5 min, and finally incubated in a cromogen solution composed by the developing buffer containing nitro blue-terazolium (NBT) (Boehringer), and 5-bromo-4chloro-3-indolyl-phosphate (BCIP) (Boehringer). The reaction was generally carried out overnight at 4°C or for 2–6 h at RT and was stopped by rinsing the sections in stop-solution (10 mM Tris–HCl pH 8, 1 mM EDTA).

2.7. Double staining In situ hybridization was carried out as described above with the following modifications. After highstringency washes in SSCT solutions, the slices were coincubated overnight at 4°C in PBST/10% FCS with the alkaline phosphatase coupled anti-DIG-Fab fragments and either the polyclonal anti-TrkB antibody (Santa Cruz-794, diluted 1:100) or the polyclonal anti ChAT antibody (Chemicon, diluted 1:200). After this incubation the sections were washed four times in PBST at RT for 10 min. Slices were then incubated in PBST/10% FCS for 3 h at RT with a biotinylated

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anti-rabbit antibody (Vector) or a biotinylated anti goat antibody (Sigma) diluted 1:200, to detect the anti-TrkB or the anti-ChAT primary antibodies, respectively. Slices were subsequently washed twice in PBST, once in PBS at RT for 10 min and then were incubated in avidine-peroxidase complex (Vector ABC Kit diluted 1:100 in PBS) at RT for 30 min. After three washes in PBS at RT for 10 min the digoxigenin labeled riboprobes were visualized through the alkaline phosphatase reaction with 4-nitro blue tetrazolium (Boehringer) and 5-bromo-4-chloro-3-indolyl-phosphate (Boehringer) in 100 mM Tris – HCl (pH 9.5), 50 mM MgCl2, 100 mM NaCl, 1 mM Levamisol (Sigma) as described in Section 2.6. Sections were then washed briefly in PBS and finally, peroxidase reaction was visualized by incubation with 0.4 mg/ml diaminobenzidine (DAB, Sigma) in PBS containing 0.5 mg/ml of glucose oxidase (Sigma). Reaction was carried out for 7 – 20 min and was stopped with two washes in PBS. Sections were mounted in water on glass slides coated with 0.5% gelatin (Merck), dried for 30 min in oven at 55°C, then were immersed 30 s in methanol, 30 s in a mixture of methanol/xylene (1:1) and finally were immersed 3 min in xylene and mounted in DPX-mount (BDH).

mRNAs known to be expressed at different levels in the brain. Fig. 1 outlines the flow-chart of the protocol used. The pairwise comparison of adjacent sections hybridized with digoxigenin-labeled probes for TrkA (Fig. 2A), MBP (Fig. 2B) or b-actin (Fig. 2C) demonstrates that under non-RF conditions (right) the signal obtained is identical to that obtained under RF conditions (left). In general, staining is seen within the first hour of development, first around the nucleus and, as the reaction proceeds, the labeling product is present over the entire cell soma. In the case of the myelin basic protein, when development is prolonged overnight, labeling can be also visualized in the processes (arrows in Fig. 2B). In contrast, with the TrkA probe the labeling remains restricted to the cell body even after prolonged development (Fig. 3C). Similarly, with the b-actin probe, labeling is usually restricted to the cell body but in a small number of hippocampal neurons, it may extend into the proximal dendrites (Fig. 3D). The nucleus is usually devoid of staining but occasionally, it may be masked by the deposit of the alkaline-phos-

2.8. In situ hybridization on fresh frozen brain slices After deep anesthesia obtained as described above, brains from adult rats were extracted, placed in embedding medium and rapidly frozen in liquid nitrogen. Slices 30 mm thick were cut at the cryostat (MicromHM 500), transferred onto poly-L-lysine coated glass slides and allowed to dry at RT for 30 min. Slices were fixed in 4% paraformaldehyde 15 min at RT and briefly washed twice in PBS. Prehybridization was carried out in a humid chamber for 90 min with prehybridization solution as described above, then prehybridization solution was replaced with the hybridization solution described above, containing 50 ng/ml of digoxigenin labeled b-actin riboprobe. Posthybridization and detection were carried out as described in Section 2.6.

3. Results

3.1. Comparison of RNAse-free with non-RNAse-free conditions In situ hybridization is usually carried out under RNAse-free conditions. To test if omitting all precautions necessary to obtain RNAse-free conditions would reduce or completely abolish the in situ signal, adult rat brain sections were probed by in situ hybridization either under RNAse-free (RF) or nominally nonRNAse-free (non-RF) conditions with riboprobes for

Fig. 1. Flow chart summarizing the experimental protocol.

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Fig. 2. Comparison of the in situ hybridization signal on coronal sections under RNAse-free (RF, left) or non-RNAse-free conditions (non-RF, right). Bright field micrographs under Nomarski optics. (A) Adult rat brain sections of basal forebrain hybridized with digoxigenin-labeled riboprobes for TrkA, showing labeling of diagonal band neurons. (B) High-power micrographs of hippocampal oligodendrocytes from adult rat brain labeled with a digoxigenin-labeled probe for myelin basic protein, arrows point at labeling in the processes. (C) Adult rat brain coronal sections of hippocampus labeled with a digoxigenin-labeled probe for b-actin. Scale bar is 100 mm for (A), 20 mm for (B) and 200 mm for (C).

phatase reaction product in heavily stained cells, especially when development is carried out overnight (compare the two cells in Fig. 3D). The specificity of the staining for TrkA in the basal forebrain was demonstrated by two criteria: (1) the lack of staining in cells outside the diagonal band (Fig. 2A) septum and caudato-putamen nuclei (not shown), in accordance to the previous literature (Holtzman et al., 1992), and (2) by the complete absence of staining with the sense riboprobe (Fig. 3A). Similarly, specificity of the staining for the MBP and the b-actin riboprobes was demonstrated by the lack of staining with the sense riboprobe (data not shown and Fig. 3B, respectively). To test if the strong paraformaldehyde fixation was responsible for the protection of the target mRNA from the risk of RNAse degradation, in situ hybridization with the b-actin probe was carried out on slices from a fresh frozen brain postfixed only 15 min in 4%

paraformaldehyde, washed twice in PBST and then prehybridized without further pretreatment. With this simple protocol the cells morphology is greatly affected but the labeling signal intensity and distribution are comparable to that obtained with the normal protocol (not shown). This demonstrates that even when the tissue is shortly fixed there is no need to carry out the in situ hybridization under RNAse free conditions.

3.2. Effects of RNAse pretreatment on the target mRNA Accidental RNAse contamination during the in situ hybridization procedure might determine the partial or total degradation of the target mRNA. To study the susceptibility to RNAse of the target mRNAs in tissues pretreated according to our protocol, slices were incubated for 30 min either at room temperature (RT, Fig.

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Fig. 3. Specificity and subcellular localization of the in situ hybridization signal. In situ hybridization on coronal sections of adult rat brain, bright field micrographs under Nomarski optics. (A) Section of basal forebrain hybridized with the TrkA sense riboprobe, no specific staining can be detected. (B) Section of the hippocampus hybridized with the sense b-actin probe, no specific staining can be detected. (C) High magnification of septal neurons stained with the anti sense riboprobe for TrkA. The staining is restricted to the cell body. (D) High magnification of hippocampal interneurons stained with the anti sense riboprobe for b-actin. One neuron displays labeling of the proximal dendrite. Scale bar is 100 mm for (A, B) and 20 mm for (C, D).

4 left) or at 37°C (Fig. 4 right) with increasing amounts of RNAse and then hybridized under non-RF conditions with the b-actin probe. The RNAse treatment was carried out at the end of all permeabilization steps, right before the prehybridization, in order to maximize penetration of the enzyme into the brain slices. At the top, control slices (C) incubated with PBS for 30 min either at RT or 37°C are shown. The simple incubation at 37°C has no detectable effect on the signal intensity with respect to RT treated slices. No apparent loss of signal can be appreciated when hippocampal slices are treated with 0.01 mg/ml of RNAse for 30 min at both temperatures. In contrast, when slices are incubated with higher RNAse concentrations, a progressively stronger decrease of the strength of the labeling is observed. At 0.1 and 1 mg/ml of RNAse the signal is markedly reduced and at 10 mg/ml of RNAse no signal can be detected. From these experiments two conclusions can be drawn: (1) endogenous RNAse does not appreciably digest the target mRNA (as already shown in Fig. 2A for TrkA and in Fig. 2B for MBP), (2) the

mRNAs contained in the slices are relatively insensitive to low amounts of exogenous RNAse, but are completely digested by high concentrations of this enzyme. Since it is unlikely that high concentrations of RNAse may enter in contact with the tissue slices through accidental contamination, we conclude that under the normal non-RF conditions used in our experiments there is no risk of significant target mRNA degradation.

3.3. Effects of the RNAse on the riboprobes The riboprobes represent the second possible substrate of the RNAse accidentally introduced during the hybridization procedure. Therefore, we tested the susceptibility of the riboprobes to an RNAse contamination during an overnight incubation at 55°C in hybridization solution. The tissue slices were omitted in these experiments. The digoxigenin-labeled riboprobe for TrkA when run on a denaturing agarose gel is composed of a main band of 460 nucleotides and a

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Fig. 4. Effects of RNAse incubation of hippocampal slices for 30 min at room temperature (RT, left) or 37°C (right) at the indicated concentrations (mg/ml). Bright field Nomarski micrographs of adult rat brain coronal sections of hippocampus labeled with a digoxigenin-labeled probe for b-actin. Controls (C) were incubated with PBS alone. Scale bar: 250 mm.

smear containing most likely short digoxigenin-labeled cRNAs fragments generated during the riboprobe synthesis (Fig. 5, lane 1C). Overnight incubation at 55°C of 100 ng of TrkA riboprobe with 0.01 mg/ml RNAse in a hybridization solution without formamide, leads to the degradation only of the riboprobe smear without decreasing the intensity of the main band (Fig. 5, lane 2, H2O—0.01). An overnight incubation with 1 mg/ml RNAse causes a partial degradation of both the riboprobe and the smear, leading to the formation of two discrete bands of lower molecular weight (Fig. 5, lane 3, H2O —1). When the TrkA riboprobe is incubated

overnight at 55°C with 0.01 mg/ml RNAse in a hybridization solution containing 50% formamide, there is no sign of degradation (Fig. 5, lane 4, 50% F—0.01) and with 1 mg/ml RNAse only a very mild decrease of both main band and smear, can be observed (Fig. 5, lane 5, 50% F—1). Taken together, these results demonstrate that formamide is able to protect the riboprobe from RNAse degradation. Remarkably, with 0.01 mg/ml RNAse there is no riboprobe degradation even in water. Whether the digoxigenin itself has or not a protective effect on the RNA, remains to be determined.

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Fig. 5. Effects of RNAse incubation on riboprobes stability. A 100-ng aliquot of digoxigenin labeled TrkA riboprobe was incubated overnight at 55°C with the indicated concentrations of RNAse (0.01 or 1 mg/ml) in hybridization solutions without (H2O) or with formamide (50% F). Probe degradation occurs only in absence of formamide at the higher RNAse concentration.

3.4. Combination of in situ hybridization with immunohistochemistry It is often desirable to combine in situ hybridization with immunohistochemistry to study coexpression with other relevant molecules. Long paraformaldehyde fixation times, such as those used in the present protocol, may have the unwanted effect to mask the epitopes recognized by the antibodies preventing therefore, the binding of the antibodies. To test the accessibility to immunohistochemistry after the in situ hybridization, slices were reacted either with antibodies against one

extracellular epitope, the TrkB high affinity receptor for the neurotrophins BDNF and NT4 (Berkemeier et al., 1991; Soppet et al., 1991), or one intracellular epitope, the enzyme choline acetyl transferase (ChAT) (Cuello and Sofroniew, 1984). For this test, a double staining protocol has been established in which incubation with the antibodies was performed after the in situ hybridization. The inverse procedure, performing the immunohistochemical labeling before the in situ hybridization greatly affected the in situ hybridization signal, possibly due to the presence of high levels of RNAse in the normal fetal serum used during the antibody incubation (data not shown). Fig. 6A, shows neurons of postnatal day 4 (P4) rat basal forebrain labeled by in situ hybridization with the probe against TrkA (blue-black alkaline phosphatase product) combined with immunohistochemistry with a polyclonal antibody against the full length isoform of the TrkB receptor (light-brown DAB–horseradish peroxidase product). The peroxidase reaction was preferentially developed using glucose oxidase as H2O2 donor since reaction with H2O2 gave an unacceptable high background. Fig. 6A shows eight labeled diagonal band neurons, out of which four are labeled only by the TrkA in situ hybridization (asterisks) and three are labeled only by the TrkB antibody (crosses). The only neuron expressing both Trk-receptors is clearly distinguishable from the other stained neurons since it displays a much darker labeling. It is worth noting that the processes (arrow), which according to their shape are, bona fide, dendrites, are only labeled by the anti

Fig. 6. Combination of in situ hybridization with immunohistochemistry. High-power Nomarski micrographs of rat basal forebrain coronal sections labeled at postnatal day 4. (A) Diagonal band neurons hybridized with a digoxigenin-labeled probe for TrkA (blue-black alkaline phosphatase reaction product, *) combined with histochemistry with anti-TrkB antibody (light-brown DAB – horseradish peroxidase, + ). The apical dendrite of a double stained neuron (dark brown labeling) is devoid of any TrkA mRNA (arrow). (B) Diagonal band neurons hybridized with a digoxigenin-labeled probe for the full length isoform of TrkB (* blue-black alkaline phosphatase reaction product of the in situ hybridization) combined with histochemistry with anti-ChAT antibody (light-brown DAB – horseradish peroxidase reaction product). Arrowheads point at dendritic varicosities of a neuron were TrkB mRNA and ChAT protein are coexpressed (dark brown labeling). Scale bar: 20 mm.

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TrkB antibody and do not contain TrkA mRNA. Fig. 6B shows basal forebrain neurons of the diagonal band hybridized with a probe for TrkB (blue-black alkaline phosphatase product) combined with histochemistry with a polyclonal antibody against ChAT (light-brown DAB–horseradish peroxidase product). Out of five labeled neurons, shown in this picture, three are labeled only by TrkB in situ hybridization (asterisks), one only by the anti ChAT antibody (cross), and one has a double label (dark brown labeled neuron). In contrast to TrkA, TrkB mRNA is localized also in the dendrites (Tongiorgi et al., 1997) and therefore in this cell also the processes are double stained and appear dark brown labeled (arrowheads). Thus, also fine aspects of the mRNA staining, related to their subcellular localization can be demonstrated by this technique, even when combined with immunohistochemistry. Specificity of the antibody staining has been tested by omitting the primary antibody after in situ hybridization (not shown), while the specificity of the in situ hybridization has been tested in previous experiments as described above. These experiments demonstrate that the two colors are clearly recognizable and, in the case of colocalization, the two colors mix giving rise to a dark-brown staining. Typically, the dark-brown double labeling covers the cytoplasm in the cell body while the region corresponding to the nucleus is devoid of the in situ labeling and is generally labeled only by the lightbrown DAB–horseradish peroxidase reaction product (Fig. 6A).

4. Discussion

4.1. In situ hybridization in a non-RNAse-free en6ironment In situ hybridization is usually carried out under RNAse-free conditions. This study demonstrates that the risk of a loss of signal due to endogenous RNAse activity or RNAse contamination has been largely overestimated. The conclusions reached by our study are threefold, in particular, we demonstrate that: (1) endogenous RNAse activity in fixed rat brain tissue is negligible, (2) to be really disruptive, a contamination from an external source should introduce into the solutions used during pretreatment at least 0.1 mg/ml of RNAse, a concentration that is unlikely to be reached through accidental contamination, (3) in hybridization solutions containing 50% formamide, the RNAse, even at high concentrations, is inactivated. Indeed, with the in situ hybridization protocol described in this study, we have obtained strong labeling also omitting all the precautions necessary to obtain RNAse-free conditions. In particular, during this procedure: (1) DEPC-treated ultrapure water was replaced by untreated ultrapure

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water, (2) all glass- and plasticware was washed with a neutral cleaning agent, rinsed in ultrapure water and used without being autoclaved. Accordingly, glass and plasticware could be washed and reused for another in situ experiment without noticing any difference among experiments, (3) none of the reagents were autoclaved for more than 20 min and were never prepared with DEPC-treated ultrapure water, (4) pipette-tips were sterilized by autoclaving only 20 min, (5) experiments were carried out with bare hands. Gloves were used exclusively when toxic reagents were used, to protect the experimenter. Paraformaldehyde is considered to be an inhibitor of the RNAse activity. Our protocol is characterized by a strong fixation of the tissue including, first, a perfusion of the animal with 4% paraformaldehyde in PBS, followed by a long postfixation of the brains (from overnight to several months) in the same fixative. While this long fixation surely concurs to the perfect preservation of the cellular morphology, allowing a very precise visualization of the cell’s processes, it is unlikely to be strictly necessary to protect the target mRNAs from digestion by endogenous RNAse. Indeed, the strong signal obtained in non-RNAse-free situ hybridization carried out after a short fixation protocol demonstrates that even a short fixation may already be effective in neutralizing the endogenous RNAse activity. On the other hand, a strong fixation in paraformaldehyde was not able to protect the target mRNAs from concentrations of exogenous RNAse above 0.1 mg/ml. Thus, tissue fixation protects mRNAs from the endogenous but not from the exogenous RNAse. The denaturing effect of formaldehyde on the enzymes is largely known (Zacks and Klibanov, 1985; Almarsson and Klibanov, 1996). Strong hydrophilic organic solvents such as formamide remove radically the hydrating water from the enzyme, causing a drastic alteration of the enzyme’s conformation, destroying therefore its enzymatic activity (Klibanov, 1989). It is therefore to be expected that no RNAse, as well as any other enzymatic activity can survive in an environment containing 50% formamide at a temperature (55°C) which is already close to the temperature of protein denaturation. This general biochemical assumption was confirmed by our direct experience demonstrating that in 50% formamide, RNAse has no or very poor enzymatic activity, even at the highest concentration tested. In conclusion, this study demonstrates that in situ hybridization can be performed without an RNAse-free environment, provided that the concentration of RNAse introduced through an accidental contamination would not reach 0.1 mg/ml. Moreover, we provide evidence that the only step sensitive to RNAse degradation is the pretreatment and that there is no risk of degradation during the hybridization step since formamide exerts a very efficient protective effect. Finally,

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our data suggest that endogenous RNAse activity might be readily neutralized through fixation.

4.2. Technical considerations on the method This method was developed with digoxigenin labeled riboprobes and was equally efficient with biotin labeled riboprobes. A non-radioactive in situ hybridization technique has several advantages with respect to the radioactive technique, including the long half life of the probes (no loss of activity in one year storage at − 20°C), the higher spatial resolution, the shorter duration of each experiment (24 – 48 h) and does not need any special safety measures or radioisotope disposal. We recommend to synthesize the riboprobes under RNAse-free conditions. The bacterial cultures from which the plasmids are isolated represent a major source of RNAse and therefore the elimination of any residual RNAse contamination from the template is a crucial requisite in order to obtain the synthesis of a reasonable amount of riboprobe. In our protocol, tissue permeabilization represent an important issue since slices are strongly fixed. To ensure a more uniform accessibility to both riboprobes and antibody we recommend the use of free floating sections and of agents such as sodium-metaperiodate and sodium-borohydride. The treatment with these agents also reduces the variability of the effects of the proteinase K treatment and allows to perform the proteolytic digestion at room temperature for times not longer than 20 min. The possibility to control the proteolytic treatment resulted particularly useful when we performed studies aiming at characterizing the subcellular localization of the mRNAs where the perfect preservation of the cell processes morphology was an important requisite. In summary, the method described here has the advantages of the non-radioactive in situ hybridization methods, i.e. is simple, rapid, flexible and has a high resolution. In addition to this, we demonstrate that under the non-RF conditions used in our experiments there is no risk of significant target mRNA or riboprobe degradation.

Acknowledgements The authors are grateful to those quoted in the text for kindly providing the plasmids for the probe synthesis. ET acknowledges a postdoctoral fellowship from the International Centre for Engineering and Biotechnology (I.C.G.E.B.) of Trieste, Italy. This work was supported by a research grant from Human Frontier Science Project Organization (HFSPO) (RG93-93) to AC.

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