Scott YoungNational Institute of Mental Health and
Éva MezeyNational Institute of Neurological Disorders and Stroke
National Institutes of Health
Bethesda, Maryland 20892
http://intramural.nimh.nih.gov/lcmr/snge/Protocols/ISHH/ISHH.html
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 probes, and performing northern analyses using these probes. RNA
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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
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
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. The slides are washed with four 15-min washes of 55° C 1X SSPE/1mM DTT followed by two 5-min washes in room temperature 1X SSPE/1mM DTT.
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:
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.
HYBRIDIZATION HISTOCHEMISTRY WITH RNA PROBES
Materials
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.
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.
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
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.
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 (
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