Section on Neural Gene Expression

Hybridization Histochemistry

Scott Young
National Institute of Mental Health and
Éva Mezey
National Institute of Neurological Disorders and Stroke
National Institutes of Health
Bethesda, Maryland 20892

Expression of genes is manifested by the production of RNA transcripts within cells. Hybridization histochemistry (in situ hybridization) permits localization of these transcripts with cellular or greater resolution (see example photomicrograph). Furthermore, the relative amounts of transcripts detected within different tissues or the same tissues under different states (e.g., physiological or developmental) may be quantified.

Basic Protocol 1 describes the preparation of the tissues from animals or humans for hybridization histochemistry. Basic Protocols 2 and 3 then describe the hybridization histochemical technique using either oligonucleotide or RNA probes (riboprobes), respectively. Basic Protocol 4 presents the use of probes labeled with digoxigenin for colorimetric detection of RNA transcripts. Basic Protocol 5 concerns the autoradiographic detection of radiolabeled probes. Support protocols provide methods for labeling oligonucleotide and RNA probes, and performing northern analyses using these probes.

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Basic Protocol 1:
PREPARATION OF TISSUES FOR HYBRIDIZATION HISTOCHEMISTRY

Our experience has shown that extensive fixation by perfusion to preserve morphology and subsequent treatment with proteases and HCl to provide access to the transcripts is not necessary unless paraffin-embedded sections are used, and may even increase unwanted background. Presented below is a basic approach involving post-fixation and treatment with acetic anhydride, ethanol and chloroform.

Materials

Lipshaw M-1 embedding matrix
Gelatin subbed, silanized or positively charged slides (see recipe; we prefer Superfrost® Plus slides)
Large staining dish and rack to hold slides
Slides frosted at one end
DEPC-treated H₂O
3.7% formaldehyde solution
Phosphate-buffered saline (PBS, see recipe)
0.25% acetic anhydride solution (see recipe)
70, 80, 95 and 100% ethanol
Chloroform

Preparation of tissue sections

1. Tissues specimens that are of appropriate size for the investigators cryostat microtome are frozen on powdered dry ice on a specimen holder with about 1ml of embedding matrix. Once the bottom of the specimen has begun to freeze, the whole specimen is covered with the powdered dry ice.

The tissue specimens may be frozen on coins with the embedding matrix. The specimens are then easily removed and stored indefinitely at -80C in an air-tight container with some ice to prevent dehydration. This approach conserves specimen holders. When the specimen is to be sectioned, it is simply frozen to a holder with some more embedding matrix. If the specimen is to be saved after cutting some sections, it can be removed from the holder by contacting the undersurface of the specimen holder with warm water until the specimen detaches.

2. Sections are then cut (e.g., 12 µ) at approximately -18° C (may need optimization for different tissue types) and either thaw-mounted onto cold, subbed slides or lifted from the knife with room temperature subbed slides. The slides with the sections are placed on a slide warmer at 42° C and after the sections have dried (about 1 min), the slides are placed at -20° or -80° C until needed.

We use talc-less gloves to avoid getting talc particles on the slides and tissue sections which can adsorb radiolabeled probe and produce spots on the film and nuclear emulsions. Certain fixatives, e.g. Histochoice® , may require polylysine-coated slides.

Prehybridization tissue preparation

3. Slides with the sections are removed from the freezer and placed on a clean surface (e.g., aluminum foil) for 10 min at room temperature.
4. Place slides in a rack and place in 4% formaldehyde solution for 5 min. Rinse twice with 1X PBS.

Small numbers of slides may be processed in DEPC-treated and autoclaved teflon coplin jars. Large numbers of slides are processed in stainless steel racks and tubs that have been DEPC-treated and autoclaved.

5. Place in fresh 0.25% acetic anhydride solution for 10 min.
6. Transfer through 70% ethanol for 1 min., 80% ethanol for 1 min., 95% ethanol for 2 min., 100% ethanol for 1 min., chloroform for 5 min., 100% ethanol for 1 min., and 95% ethanol for 1 min. Remove the rack from the ethanol and allow to air dry (with ethanol draining toward the frosted end of the slide).

The protocols in this unit are easily applied to tissue cultures and cytospin samples, as well. Tissue cultures should be grown on glass slides with removable chambers and then fixed and treated on those slides because plastic slides and the chambers are dissolved by the chloroform. Similarly, cell suspensions may be deposited by cytospinning the samples onto subbed slides. Chloroform treatment is not necessary for riboprobes, but hasn't been studied recently for oligoprobes.

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Basic Protocol 2:
HYBRIDIZATION HISTOCHEMISTRY WITH OLIGODEOXYNUCLEOTIDE PROBES

This protocol can be used when less sensitivity is needed than is provided by RNA probes (Basic Protocol 3). Also, less molecular biological expertise is necessary and is a good initial approach to hybridization histochemistry.

Materials

20X SSPE (see recipe)
Formamide
Dithiothreitol (DTT)
Hybridization solution (see recipe)
Glass coverslips
Nunc Sterile Bio-assay dishes (245 x 245 x 30 mm)
Whatman 3MM chromatography paper

Hybridization and washes of the sections
1. Cover the inside of the Bio-assay dish top (it has a larger surface area than the bottom) with a piece of chromatography paper. Wet with 50% formamide/4x SSPE and lay the slides with the sections up on the paper.
2. Place hybridization solution containing about 1 x 10⁶ dpm ³⁵S-labeled (or 1-5µl digoxigenin-labeled) oligodeoxynucleotide /50µl hybridization solution onto the tissue section and cover with a coverslip

45 µl of hybridization solution is enough to cover two adult rat coronal sections under a 18 x 30mm coverslip. The amount of hybridization solution used should be scaled up proportionately if a larger coverslip is used to cover a larger tissue section. We have found no need to pretreat our coverslips, but they should be dust free.

3. Cover the slides with the bottom of the Bio-assay dish and place in a 37° C incubator for 20-24 hr.
4. Place slides with frosted ends up into a staining dish containing 1X SSPE/1mM DTT and gently slide the coverslip until a bit overhangs the slide. The coverslip is then pried off with forceps. Do not allow the section to dry until step 7.
5. The slide is then placed in a rack in tub with 1X SSPE/1mM DTT until all the slides have had their coverslips removed and been placed in the same rack.
6. Rinse the slides 4 times 5 min. with 1X SSPE/1mM DTT at room temperature with gentle shaking at 50rpm.
7. Wash the slides next with two 30-min washes of 55° C 1X SSPE/1mM DTT followed by two 5-min washes in room temperature 1X SSPE to cool.

At this point, if the slides are to be processed for digoxigenin-labeled probes, proceed to the Basic Protocol 4.

7. The slides are then rapidly dipped into H₂O and then 70% ethanol and blown dry while the slides are oriented with their frosted ends down.

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Basic Protocol 3:

HYBRIDIZATION HISTOCHEMISTRY WITH RNA PROBES
RNA probes offer the greatest sensitivity for hybridization histochemistry. This can be important in initial mapping surveys so that the results are as inclusive as possible.

Materials

20X SSPE (see recipe)
Formamide
Dithiothreitol (DTT)
Hybridization solution (see recipe)
Glass coverslips
Nunc Sterile Bio-assay dishes (245 x 245 x 30 mm)
Whatman 3MM chromatography paper
RNase A solution (see recipe)

Hybridization and washes of the sections
1. Cover the inside of the Bio-assay dish top (it has a higher surface area than the bottom) with a piece of chromatography paper. Wet with 50% formamide/4xSSPE and lay the slides with the sections on the paper.
2. Place hybridization solution containing about 1 x 10⁶ dpm ³⁵S-labeled (or 1-5µl digoxigenin-labeled) riboprobe/50µl hybridization solution onto the tissue section and cover with a coverslip

45µl of hybridization solution is enough to cover two adult rat coronal sections under a 18 x 30mm coverslip. The amount of hybridization solution used should be scaled up proportionately if a larger coverslip is used to cover a larger tissue section. We have found no need to pretreat the coverslips, but they should be dust free. If background is a problem, consider using siliconized coverslips.

3. Cover the slides with the bottom of the Bio-assay dish and place in a 55° C incubator for 20-24 hr. Occasionally, increasing the hybridization temperature up to 65° C reduces 'stubborn' background.
4. Place slides with frosted ends up into a staining dish containing 1X SSPE/1mM DTT and gently slide the coverslip until a bit overhangs the slide. The coverslip is then pried off with forceps. Do not allow the section to dry until step 9.
5. The slide then is placed in a rack in tub with 4X SSPE/1mM DTT until all the slides have had their coverslips removed and been placed in the same rack.
6. Rinse the slides 4 times 5 min. with 4X SSPE/1mM DTT at room temperature with gentle shaking at 50rpm.
7. Incubate the slides in the RNase solution at 37° C for 30 min. Rinse twice for 5 min in 0.1X SSPE/1mM DTT at room temperature.
8. Wash the slides next with two 30-min washes of 65° C 0.1X SSPE/1mM DTT followed by two 5-min washes in room temperature 1X SSPE to cool.

At this point, if the slides are to be processed for digoxigenin-labeled probes, proceed to the Basic Protocol 4.

9. The slides are then rapidly dipped into H₂O and then 70% ethanol and blown dry while the slides are oriented with their frosted ends down.

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Basic Protocol 4:
DETECTION OF DIGOXIGENIN-LABELED PROBES

Digoxigenin-labeled probes are detected by using antibodies directed toward the digoxigenin moiety. These primary antibodies are usually conjugated to alkaline phosphatase (AP) or horseradish peroxidase (HRP) which deposit a colored reaction product in the presence of the appropriate substrate at the site of hybridized probe. Furthermore, the HRP-conjugated antibody may be used in a further amplification scheme involving biotinyl tyramide and subsequent detection with streptavidin-conjugated chromophore or enzyme (e.g., AP or HRP). This is referred to as Tyramide Signal Amplification (TSA).

Direct detection of digoxigenin

Materials

20X SSPE (see recipe)
Buffer 1 (100mM Tris-HCl, 150mM NaCl, pH7.5 at room temperature)
Buffer 3 (100mM Tris-HCl, 100mM NaCl, 50mM MgCl₂, pH9.5 at room temperature)
Normal goat serum (NGS, Vector)
Triton X-100
Sheep polyclonal anti-digoxigenin-AP (Boehringer Mannheim)
75mg/ml nitroblue tetrazolium chloride (NBT) in 70% dimethylformamide
50mg/ml 5-bromo-4-chloro-3-indolyl phosphate p-toluidinium salt (BCIP) in 70% dimethylformamide
Levamisole
Cytoseal 60 (Stephens Scientific)

1. Transfer slides from SSPE wash above to Buffer 1 for two 5-min washes.
2. Transfer slides to Buffer 1 with 5% NGS and 0.6% Triton X-100 for 30 min.
3. Transfer slides to Buffer 1 with 5% NGS, 0.6% Triton X-100, and 1:2000 anti-digoxigenin-AP for 5-16 hrs at room temperature with gentle rocking.
4. Transfer slides to Buffer 1 for two 10-min washes.
5. Transfer slides to Buffer 3 for 5 min.
6. Incubate several hours to overnight at room temperature in the dark in Buffer 3 with 0.34mg/ml NBT and 0.18mg/ml BCIP. Levamisole to 1mM may be added to block peripheral-type endogenous alkaline phosphatase.
7. Wash the slides with four 30-min washes in 1x SSPE.

These long washes eliminate residual NBT (and BCIP) that interacts nonspecifically with nuclear emulsion, but are not enough for Kodak emulsions, necessitating the use of Ilford emulsion.

8. Dip briefly into water and blow dry.

The slides may be incubated in slide mailers at steps 3 and 6 to conserve reagents. Slides should be thoroughly dried on a slide warmer before coverslipping (in an organic-based mountant) or proceeding to autoradiographic detection (Basic Protocol 5). If the sections are not dry before coverslipping, the signal may be lost.

9. Coverslip slides with Cytoseal 60 (or, similar organic-based) mountant if not proceeding to autoradiographic detection (Basic Protocol 5).

Tyramide Signal Amplification (TSA) detection of digoxigenin-labeled probes

This alternative procedure for digoxigenin detection uses peroxidase-conjugated anibody in an amplification scheme involving biotinyl tyramide and subsequent detection with streptavidin-conjugated fluorochromes, peroxidase or alkaline phosphatase. This approach produces an increased signal as compared to Basic Protocol 4 and also enables the researcher to develop the signal as a fluorescent product compatible with a second non-radioactive hybridization histochemical or an immunohistochemical labeling.
(see also the protocols for Immunohistochemistry)

Materials

Buffer 1 (100mM Tris-HCl, 150mM NaCl, pH7.5 at room temperature)
Buffer 2 (100mM Tris-HCl, 150mM NaCl, pH8 at room temperature)
Normal goat serum (NGS, Vector)
Triton X-100
Sheep polyclonal anti-digoxigenin-POD (Boehringer Mannheim)
Renaissance® TSA-Indirect kit (New England Nuclear) which contains:
2x Diluent
DuPont blocking reagent
Biotinyl tyramide
Streptavidin-HRP conjugate
Streptavidin-Texas-Red© (NEN)
Streptavidin-fluorescein (NEN)
Streptavidin-HRPO (NEN)
Streptavidin-alkaline phosphatase (Jackson ImmunoResearch Laboratories)
Diaminobenzidine (DAB) and urea-hydrogen peroxide tablets (Sigma)

1. Transfer slides from SSPE wash above to Buffer 1 for two 5-min washes.
2. Transfer slides to Buffer 1 with 5% NGS and 0.6% Triton X-100 for 30 min.
3. Transfer slides to Buffer 1 with 5% NGS, 0.6% Triton X-100, and 1:300-1:600 anti-digoxigenin-POD for 2 hrs with gentle rocking, and then overnight at 4° C.
4. Transfer slides to Buffer 1 for three 5-min washes.
5. Transfer slides to 1x Diluent for 5 min.
6. Transfer slides to 1x Diluent with 1:50-1:100 Biotinyl tyramide for 10 min.
7. Wash slides with three 5-min washes in Buffer 1

Detection of the labeled probe may now proceed in several ways, through the use of streptavidin-Texas red or streptavidin-fluorescein (a), streptavidin-HRP (b) or streptavidin-alkaline phosphatase conjugates (c) in increasing order of sensitivity:

8a. Prepare a 1:2000 dilution of streptavidin-Texas red or of streptavidin-fluorescein in Buffer 1. Incubate slides in this solution for 60 min at room temperature.
9a. Wash slides four times, each time by immersing 5 min in fresh Buffer 1.

8b. Prepare a 1:3000 dilution of streptavidin-HRP conjugate in Buffer 1 containing 0.5% DuPont blocking reagent. Incubate slides in this solution for 60 min at room temperature.
9b. Wash slides four times, each time by immersing 5 min in fresh Buffer 2.
10b. Dissolve one DAB tablet and one urea/hydrogen peroxide tablet in 15 ml of Buffer 2. Transfer the slides to this solution and incubate until a signal develops at about 5-10 min.

Sometimes the signal becomes apparent while looking directly at the tissue section. With some other mRNAs, the sections need to be examined microscopically.

11b. Wash the slides twice with Buffer 2 using the technique of step 9b.

8c. Prepare a 1:25,000 dilution of streptavidin-alkaline phosphatase conjugate in Buffer 1 with 5% NGS, and 0.6% Triton X-100. Incubate slides in this solution for 60 min at room temperature. Then follow steps 4-9 of Basic Protocol 4.

12. Dip slides briefly into water and blow dry. Thoroughly dry slides on a slide warmer.

Slides should be thoroughly dried on the slide warmer before mounting of coverslips; otherwise the signal may be lost.

13. Apply coverslips to slides using Cytoseal 60 or similar organic-based mounting medium if not proceeding to autoradiographic detection (Basic Protocol 5).

One can also use fluorescein-12-UTP (Boehringer Mannheim) instead of digoxigenin-labeled UTP at the same concentrations to label RNA for nonradioactive hybridization histochemistry (Support Protocol 2 and Basic Protocol 3). The tyramide signal amplification (TSA; Alternate Protocol 1) method is then used with a HRPO-conjugated sheep polyclonal anti-fluorescein antibody (Boehringer Mannheim), followed by biotinylated-tyramide and streptavidin-HRP or streptavidin-alkaline phosphatase. The biotinylated-tyramide and streptavidin-alkaline phosphatase detection is very sensitive, close to that of radiolabeled probes. Furthermore, this permits the simulataneous detection of two different transcripts by non-radioactive means. After detection of the digxigenin-labeled probe (Basic Protocol 4), proceed without drying to Alternate Protocol 1 and use the HRPO-conjugated sheep polyclonal anti-fluorescein antibody in step 2, followed by biotinylated-tyramide and streptavidin-fluorochrome or streptavidin-HRP.

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Basic Protocol 5:
DETECTION OF RADIOLABELED PROBES

Detection of radiolabeled probes entails apposition of the samples to x-ray film or phosphorimaging plates and subsequent development. Higher, cellular resolution entails coating of the sample with a nuclear emulsion (described here). These steps are performed after detection of the digoxigenin-labeled probes if the two types of probes are used simultaneously.

Materials

Ilford K5.D (necessary for digoxigenin double labeling) or Kodak NTB-3 nuclear emulsion
7.5M ammonium acetate
Black slide boxes
Desiccant capsules (e.g., Humi-caps from United Desiccants, 800-989-3374)
Kodak D-19 photographic developer
Kodak Rapid Fix (without hardener)
Cytoseal 60 (Stephens Scientific)

Prepare the nuclear emulsion for coating the sections
1. Under safelight conditions, scoop out 40 ml of emulsion with spatula into a coplin jar containing 1.6 ml of 7.5M ammonium acetate (final concentration of 300mM).
2. Place the coplin in a 40° C water bath for 20-30 mins to allow air bubbles to rise. Mix gently and look for bubbles on a clean slide after dipping it into the emulsion.
3. Dip slides into the emulsion, and stand up for several hours to dry.

We place 5 slides in red plastic slide grips and dip them 5 at a time into the emulsion. We then hang them from a custom-made plexiglass holder.

4. The emulsion-coated slides are placed in black slide boxes with desiccant capsules. Tape the edges of the box with black photography tape and store the boxes at 4° C in the dark.

Develop the emulsions and stain the tissue sections

5. Put the slides in racks and pass through the solutions as follows at 17° C. (with agitation every 30s): D-19 for 2mins; running tap water 15s with slight agitation; and Kodak Rapid Fix (without hardener) for 2mins.

The room lights may be turned on after all slides are fixed.

6. Rinse in running tap water for 8 minutes. Counterstain, if desired, for 30s in 0.4% toluidine blue, 2µg/ml ethidium bromide, hematoxylin/eosin, or stain of choice, and rinse again briefly to remove excess stain.

Some stains may obscure or destroy colorimetric detection of the digoxigenin probe or silver grains (e.g., periodic acid Schiff, cresyl violet).

7. Dip very briefly into deionized water, then 70% ethanol and place on slide warmer to thoroughly dry.
8. Coverslip slides with Cytoseal 60 (or, similar organic-based) mountant.

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REAGENT AND SOLUTIONS

Use DEPC-treated H₂O to make solutions for solutions for pretreatment and hybridization. De-ionized H₂O may be used for the subsequent wash and digoxigenin development steps.

Slides for mounting tissue sections:

1. Subbed slides
   
   • Place slides in racks and soak in soap solution for 1 hr.
     
   • Rinse in deionized water. Change the water several times to be sure that all of the soap is removed.
     
   • Dissolve 1.88 g of gelatin (300 bloom swine) in 750 ml hot H₂O (do not allow to boil). Cool and dissolve 0.188 g CrK(SO₄)₂*12H₂O in the solution.
     
   • Dip the slides into the subbing solution, drain the slides onto a paper towel and allow to air dry for 1 hr.
     
   • Dip the slides into the subbing solution again. Drain and cover loosely with plastic wrap or bench paper.
     
   • When thoroughly dry, store the slides in slide boxes.
   
   
   
2. Silanized slides:
   
   • Clean slides with a lint free cloth manually (lots of work, but needed) and put them in racks.
     
   • Dip slides in 2% aminoalkalynsilane (Sigma A-3648) in dry acetone for 10 sec.
     
   • Rinse in deionized water 3 times.
     
   • Air dry overnight and store in boxes protected from dust.
   
   
   
3. Positively charged slides:
   
   • We buy these slides: Superfrost Plus microscope slides (4951+, Erie Scientific, Portsmouth, NH 03801) or you can make them as follows:
     
   • Clean slides with a lint free cloth manually (lots of work, but needed) and put them in racks
     Dip slides in 50µg/ml poly-L-lysine
     
   • Air dry overnight and store in boxes protected from dust.

10X Phosphate-buffered saline, pH 7.4 (PBS)

90 g NaCl
1.22 g KH₂PO₄
8.15 g Na₂HPO₄
H₂O to 1 liter

3.7% Formaldehyde solution

Per liter, add 100 ml 37% formaldehyde and 100 ml 10X PBS

0.25% acetic anhydride, pH 8.0

Per 100 ml, mix 1.49 ml triethanolamine and 420 µl concentrated HCl. Then add and mix 0.25 ml acetic anhydride.
Use immediately.

20X SSPE

Dissolve 175.2 g NaCl, 27.6 g NaH₂PO₄*H₂O and 7.4 g Na₂EDTA in 800 ml H₂O. Adjust pH to 7.4 with NaOH and then volume to 1 liter with H₂O. Final [Na] of 20x SSPE=3.2M.

Ribonucleic acid solution

Dissolve salmon sperm DNA (100 µg/ml final; per ml: 250 µl of 10mg/ml stock), yeast total RNA (250 µg/ml final; per ml: 313 µl of 20mg/ml stock) and yeast tRNA (250 µg/ml final; per ml: 250 µl of 25mg/ml stock) in H₂O. Store indefintely at -20° C.

50X Denhardts solution

1 g ficoll
1 g polyvinylpyrrolidone
1 g bovine serum albumin
H₂O to 100ml
Store indefintely at -20° C.

Hybridization buffer

23.8 ml formamide
0.95 ml 1M Tris-HCl, pH 7.4
0.19 ml 250 mM EDTA, pH 8.0
3.75 ml 4M NaCl
9.52 ml dextran sulfate
0.95 ml 50X Denhardts solution
H₂O to 40ml.
Store indefintely at -20° C.

Hybridization solution

Add probe plus H₂O to 8 µl plus 4 µl ribonucleic acid solution and mix. Heat at 65° C for 5 min. and then cool rapidly on ice to room temperature. Add 84 µl hybridization buffer, 2 µl 5M DTT (5g DTT plus 2.72ml H₂O), 1 µl 10% sodium thiosulfate, and 1 µl 10% sodium dodecyl sulfate. Mix well. Scale up appropriately.

RNase A solution

Dissolve 1 ml of 10 mg/ml RNase A (e.g., Sigma R6513 dissolved to 10mg/ml in 10mM Tris-HCl, pH7.5/15mM NaCl. Stored at -20° C. No boiling to denature DNase is recommended) just prior to use in 500 ml pre-warmed (37° C) RNase buffer (62.5 ml of 4M NaCl, 2.5 ml of 2M Tris-HCl, pH8.0, and 0.5 ml of 0.25M EDTA)

We have had RNase A lots that are more potent than others, so when new RNase A is obtained, it should be tried over a range of concentrations to determine the best signal-to-noise ratio.

Northern hybridization solution

20 ml formamide
2 ml 20X SSPE
8 ml 50% Dextran Sulfate
4 ml 50X Denhardt's solution
400 µl 25 mg/ml tRNA
500 µl 20 mg/ml total yeast RNA
400 µl 10 mg/ml single-stranded DNA
400 µl 10% SDS
H₂O to 40 ml
Store indefintely at -20° C.

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COMMENTARY
Background Information
Hybridization histochemistry provides a method to detect specific mRNAs in tissue sections. Furthermore, as mRNA levels may change from one state to another (e.g., during development or after physiological manipulations), hybridization histochemistry can provide snapshots through the course of a dynamic situation. This unit reprises our protocols to examine the expression of genes within tissue sections at a light microscopic resolution (Young et al., 1986; Young et al., 1990; Bradley et al., 1992; Young, 1992). There are a number of excellent sources of information for the reader interested in localizing transcripts in whole mount tissues, to chromosomes or at the electron microscopic level (Wilkinson, 1992; Rosen and Beddington, 1993; Albertson et al., 1995; Morey, A.L., 1995; Swiger, and Tucker, 1996).
Hybridization histochemistry is generally amenable to combining with other techniques, such as immunohistochemistry, tract-tracing (Burgunder, and Young, 1988) and in vitro receptor autoradiography (Westlake et al., 1994). The combination with immunohistochemistry and/or tract-tracing may necessitate perfusion fixation of the animal (in order to preserve immunoreactivity and/or tracer deposition) prior to freezing the specimen and sectioning it. These sections, however, generally have a reduced signal-to-noise ratio for the hybridization histochemistry. The immunohistochemical steps are usually performed after the the hybridization histochemical ones to avoid loss of mRNA from exposure to RNases present in the antibody and development solutions. To use the same tissue for in vitro receptor autoradiography and hybridization histochemistry, alternate fresh-frozen sections are used.

Critical Parameters and Troubleshooting
Successful hybridization histochemistry seeks a balance between preserving tissue morphology and permeabilizing the tissue to allow access of the probe to the transcripts. While a number of protocols utilize HCl and/or proteases to permeabilize the tissue sections, our approach avoids these harsh treatments through the use of chloroform to de-fat the sections. However, paraffin-embedded tissue sections require the use of protease.
Our experience has shown that the longer riboprobes that, obviously target longer stretches of the transcripts, than single oligodeoxynucleotide probes, offer greater sensitivity. However, the use of multiple oligodeoxynucleotide probes targeted against the same transcript can significantly improve sensitivity compared to a single oligodeoxynucleotide probe. Theoretically, and in practice, riboprobes are more sensitive than the equivalent stretch of bases presented by labeled, double-stranded cDNA probes. Some researchers also employ alkaline hydrolysis of their riboprobes to increase the ease of tissue penetration. Again, we have not found this to help with our protocol and may be inconsistent in the probe sizes produced. References to other approaches and further discussion are presented at the end of the unit (Valentino et al., 1987; Wilkinson, 1992)
We label our probes for hybridization histochemistry with ³⁵S for a number of reasons. It provides greater resolution and higher efficiency of grain production than either ³²P or ³³P. Also, it has a half-life of 87 days, compared with 14 and 25 days for ³²P or ³³P, respectively. These reasons more than compensate for ³⁵S's lower specific activity. Although ³H provides greater resolution and has a much longer half-life, its specific activity is so low, that it is not practical to label probes targeted against transcripts of relatively low abundance. We generally use the digoxigenin-labeled probes to enable double simultaneous detection of two different transcripts within the same tissue sections (and within the same cells). The radiolabeled probes permit more accurate quantitation of transcript levels and are still more sensitive.
Controls for specificity are, of course, the essence of any experiment. Unfortunately, there is no single, absolute control for hybridization histochemistry. Instead, the researcher relies on as many different checks as possible. The ones we prefer, in a roughly descending order of usefulness, are as follows: 1. Same distribution of signal with probe directed against a different portion of the same transcript. 2. Blockage of signal by prior hybridization with unlabeled probe. 3. Correlation of signal with immunocytochemical results. 4. Different distribution of signal with probes against unrelated transcripts, including sense probes (be aware, however, that occasionally the sense probe detects mRNA transcribed from the opposite DNA strand. However, a signal with the sense probe does not necessarily invalidate the findings obtained with the antisense probe). 5. Northern analysis using the probe under the same degrees of stringency shows band(s) of expected size(s). Two commonly used controls are not recommended: the use of RNase prior to hybridization is analogous to using a protease prior to immunohistochemistry and the dilution of of labeled probe with unlabeled probe only serves to reduce the specific activity of the probe. Both of these procedures are essentially worthless.
One should be aware of the potential artifacts that arise from autoradiography and/or color techniques. Positive and negative chemography, the spurious creation and destruction of grains, respectively, are constant concerns with autoradiography. Positive chemography is probably more common and is best assessed using sections that were not hybridized, or hybridized with a sense probe. Grains are especially susceptible to loss during staining or after coverslipping if moisture still remains in the tissue sections. These and other aspects of autoradiography are expertly discussed by Rogers (1979). Color development artifacts with alkaline phosphatase may be due to endogenous peripheral-type enzyme and may be blocked with levamisole (intestinal alkaline phosphatase is more refractory and needs treatment with 0.1M HCl for 10 min. at room temperature; Kiyama and Emson, 1991). Also, DTT that is present during the enzymatic development can impart a B purplish color. The use of non-hybridized sections should reveal whether adventitious color formation is occurring. Loss of alkaline phosphatase staining occurs with exposure to ethanol.

Anticipated Results
Hybridization histochemistry should enable the researcher to determine whether a given gene is expressed in particular cells. Figure 1 shows the simultaneous detection of two different transcripts detected through the use of radiolabeled and digoxigenin-labeled probes. Riboprobes are more sensitive than oligodeoxynucleotide probes, enabling one to see 5 or fewer transcripts per cell, and traditionally, ³⁵S-labeled probes are more sensitive than colorimetrically detected ones. However, as we gain more experience with amplification techniques, this superiority of radiolabeled probes may vanish. Furthermore, the recent introduction of the tyramide amplification system (TSA, also known as catalyzed reporter deposition or CARD) (Bobrow et al., 1989) offers a number of branch points for varying degrees of amplification and different reaction products (Kerstens et al., 1995; Hunyady et al., 1996).

Quantitative Analysis of Autoradiograms
X-ray or tritium sensitive films may provide the easiest means to quantitation if the signal is sufficient and the cells are closely grouped. In these cases, the film optical densities can be converted to copies of probe hybridized through the use of simultaneously exposed brain paste standards that incorporate known amounts of the radioisotope. Phosphorimaging devices (e.g., those of Fuji Medical Systems or Molecular Dynamics) offer 2 advantages over films: their sensitivity is up to 40-fold greater and the signals are directly proportional to the amount of hybridized radiolabeled probe. We generally examine our sections with the phosphorimaging system prior to dipping them into nuclear emulsion. Detailed protocols for quantitative analysis of autoradiograms are available (Gerfen, 1989; Young, 1992).

Time Considerations
Hybridization histochemistry may be viewed as composed of three steps: preparation of the tissue sections, hybridization and washing of the sections, and detection of the hybridization signal. Preparation of the tissue sections, after collection of the tissue specimens, essentially consists of cutting the sections and, of course, depends upon the numbers of sections needed and the size of the region(s) studied. This may take hours to weeks.
The hybridization and washing steps take either 2 or 3 consecutive days, depending on whether radiolabeled or digoxigenin-labeled probes are used, respectively. Detection of the digoxigenin-labeled probes is then complete at the end of the third day. Depending on the signal strength and degree of resolution needed, radiolabeled probe deposition can be determined over the course of minutes using film or phosphorimaging plates to months after coating with nuclear emulsion.

Literature Cited

1. Albertson, D.G., Fishpool, R.M. and Birchall, P.S. 1995. Fluorescence in situ hybridization for the detection of DNA and RNA.Methods Cell Biol 48:339-364.
   
2. Bobrow, M.N., Harris, T.D., Shaughnessy, K.J. and Litt, G.J. 1989. Catalyzed reporter deposition, a novel method of signal amplification.J Immun. Meth. 125:279-285.
   
3. Bradley, D.J., Towle, H.C. and Young, W.S. III. 1992. Spatial and temporal expression of alpha and beta thyroid hormone receptor mRNAs, including the b₂ subtype, in the developing mammalian nervous system.J. Neurosci. 12:2288-2302.
   
4. Burgunder, J.-M. and Young, W.S. III. 1988. The distribution of thalamic projection neurons containing cholecystokinin messenger RNA, using in situ hybridization histochemistry and retrograde labeling.Mol. Brain Res. 4:179-189.
   
5. Gerfen, C.R. 1989. Quantification of in situ hybridization histochemistry for analysis of brain function. In Methods in Neuroscience (Conn, P.M., ed) pp. 79-97. Academic Press, New York
   
6. Hunyady, B., Krempels, K., Harta, G. and Mezey, É . 1996. Immunohistochemical signal amplification by catalyzed reporter deposition and its application in double immunostainings.J. Histochem. Cytochem.12:1353-1362
   
7. Kerstens, H.M.J., Poddihe, P.J. and Hanselaar, A.G.J.M. 1995. A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine.J. Histochem. Cytochem. 43:347-352.
   
8. Kiyama, H. and Emson, P.C. 1991 An in situ hybridization histochemistry method for the use of alkaline phosphatase-labeled oligonucleotide probes in small intestine. J. Histochem. Cytochem. 39: 1377-1384.
   
9. Morey, A.L. 1995. Non-isotopic in situ hybridization at the ultrastructural level.J. Pathol. 176:113-121.
   
10. Rogers, A.W. 1979. Techniques of Autoradiography. Elsevier, New York.
    
11. Rosen, B. and Beddington, R.S. 1993. Whole-mount in situ hybridization in the mouse embryo: gene expression in three dimensions.Trends Genet. 9:162-167.
    
12. Swiger, R.R. and Tucker, J.D. 1996. Fluorescence in situ hybridization: a brief review.Environment. Mol. Mutagen. 27:245-254.
    
13. Valentino, K.L., Eberwine, J.H. and Barchas, J.D. (eds.). 1987. In Situ Hybridization. Applications to Neurobiology. Oxford University Press, New York.
    
14. Westlake, T.M., Howlett, A.C., Bonner, T.I., Matsuda, L.A. and Herkenham, M. 1994. Cannabinoid receptor binding and messenger RNA expression in human brain: an in vitro receptor autoradiography and in situ hybridization histochemistry study of normal aged and Alzheimer's brains.Neuroscience 63:637-652.
    
15. Wilkinson, D.G. (ed). 1992. In Situ Hybridization. A Practical Approach. Oxford University Press, New York.
    
16. Young, W.S. III. 1992. In situ hybridization with oligodeoxyribonucleotide probes. In In Situ Hybridization. A Practical Approach (Wilkinson, D.G., ed) pp. 33-44. Oxford University Press, New York
    
17. Young, W.S. III., Mezey, É . and Siegel, R.E. 1986. Vasopressin and oxytocin mRNAs in adrenalectomized and Brattleboro rats: analysis by quantitative in situ hybridization histochemistry.Mol. Brain Res. 1:231-241.
    
18. Young, W.S. III., Reynolds, K., Shepard, E.A., Gainer, H. and Castel, M. 1990. Cell-specific expression of the rat oxytocin gene in transgenic mice. J. Neuroendocrinol.2:917-925.

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Photomicrograph shows oxytocin and vasopressin neurons within the human supraoptic nucleus labeled with 35S-labeled and digoxigenin-labeled probes, respectively. Neurons containing the digoxigenin-labeled probe show a dark stain from development of the alkaline phosphatase on the anti-digoxigenin antibodies. The deposition of the radiolabeled probe is indicated by the green-colored silver grains. Click on the image to see an enlarged version.

The Section on Neural Gene Expression is in the Laboratory of Cellular and Molecular Regulation, Division of Intramural Research Programs is within the National Institute of Mental Health (NIMH) is a part the National Institutes of Health (NIH), and is a component of the U.S. Department of Health and Human Services.

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