ProtocolSimultaneous isotopic and nonisotopic in situ hybridization histochemistry with cRNA probes
Section snippets
Type of research
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Coexpression studies: Determining colocalization of two distinct populations of mRNA in single cells (e.g., [11])
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Localization of mRNAs to specific cell populations (e.g., 6, 11).
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Generation of cRNA probes for in situ hybridization histochemistry.
Time required
Overall=1–4 months (depending on the exposure times required for radiolabeled hybrids).
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Creation of templates using PCR=3–4 days
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Preparation of frozen tissue sections on poly-l-lysine coated slides=1 day
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Generation of probes labeled with Digoxigenin-UTP=6 h
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Generation of probes labeled with h
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Hybridization=1 day (prehybridization=2–3 h; hybridization=4 h; post-hybridization=3 h)
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Visualization of digoxigenin tagged hybrids=1–2 days (immunohistochemistry=4–6 h; alkaline phosphatase
Materials
- 1.
Tissue
- 1.1.
Fresh frozen tissue, stored at −70°C
- 1.1.
- 2.
Equipment
- 2.1.
Thermal Cycler
- 2.2.
Horizontal gel electrophoresis system
- 2.3.
Ultraviolet transilluminator
- 2.4.
Oven
- 2.5.
Cryostat
- 2.6.
Beta scintillation counter
- 2.7.
Wheaton brand borosilicate staining dishes and racks (Wheaton Scientific Products)
- 2.8.
Hybridization chambers (e.g., Bioassay trays, Nunc) with sink matting
- 2.9.
Heating block
- 2.10.
2 Shaking water baths
- 2.11.
Shaking table
- 2.12.
Hem-tek slide staining set (Baxter Scientific Products)
- 2.13.
Slide grips (Polysciences)
- 2.14.
Light tight box to suspend the slides
- 2.1.
- 3.
Chemicals and
General laboratory procedures
Reagents used for this protocol should be of high or molecular biology grade. All solutions and equipment used for the steps preceding the post-hybridization wash must be RNase-free. We routinely treat all solutions with DEPC. We add 1 ml DEPC per liter solution, let stir in a hood for 2 h and autoclave to inactivate the DEPC. An exception is Tris–HCl which is made directly with DEPC-treated H2O. All glassware and metal equipment are baked at 200°C for at least 6 h and sterile disposable
Results
A typical result obtained with the procedure described above is shown in Fig. 3 (Film autoradiography) and Fig. 4 (Emulsion autoradiography). Using a digoxigenin tagged cRNA probe we consistently obtained signals for a transcript of medium abundance, for example, preprotachykinin mRNA in striatal neurons 6, 11; attempts by us and others to use digoxigenin labeled oligonucleotides failed in dual label studies, presumably because of the lower sensitivity of oligonucleotide probes [12]. Given the
Generation of templates using PCR
If no bands can be observed on ethidium bromide stained agarose gels following the first round of amplification (using the outer primer pair) we often proceed to the second round using inner primer pair reasoning that the amplification product may be present in low abundance escaping detecting by ethidium bromide staining. If no product is obtained in the second round of amplification we consider a number of possibilities. The target may be absent from or degraded in the starting material
Quick procedure
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Create linear templates for run-off transcription of cRNA-probes using PCR.
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Prepare sections of fresh frozen brain by cryosectioning.
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Transcribe digoxigenin- and cRNA probes.
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Hybridize with a digoxigenin-tagged and a cRNA probe simultaneously.
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Visualize the mRNA-digoxigenin-cRNA hybrids immunohistochemically.
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Visualize the radioactively-labeled hybrids by autoradiography.
Essential literature references
Refs. 1, 2, 6, 9, 11.
Acknowledgements
Supported by USPHS grants NS31579, NS34361, AG11337, and DFG-grant SFB505.
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