Elsevier

Brain Research Protocols

Volume 3, Issue 1, September 1998, Pages 22-32
Brain Research Protocols

Protocol
Simultaneous isotopic and nonisotopic in situ hybridization histochemistry with cRNA probes

https://doi.org/10.1016/S1385-299X(98)00017-8Get rights and content

Abstract

In situ hybridization histochemistry is widely used to study gene expression at the mRNA level in tissues and cells. Double label in situ hybridization allows for coexpression studies. We describe a protocol for the simultaneous hybridization of two cRNA probes tagged with 35S-CTP and digoxigenin-UTP, respectively, to frozen brain tissue sections. Hybridization signals of digoxigenin-tagged probes appear as purple cytoplasmic staining following detection of digoxigenin residues by an alkaline-phosphatase-(AP)-linked antibody. Signals resulting from hybridization of radiolabeled probes are detected as silver grains overlying cellular profiles in sections coated with autoradiographic emulsion. Grain counting allows for semiquantitatively estimates of the cellular expression levels of transcripts. Suitable cRNA-probes can be derived from linear templates generated by polymerase chain reaction (PCR) using nested primers which contain RNA-polymerase promotor sites. The cRNA-probes are sensitive and allow an application of this protocol to the detection of a wide range of mRNAs of medium or low abundance.
Themes: Cellular and molecular biology
Topics: Staining, tracing, and imaging techniques

Section snippets

Type of research

  • Coexpression studies: Determining colocalization of two distinct populations of mRNA in single cells (e.g., [11])

  • Localization of mRNAs to specific cell populations (e.g., 6, 11).

  • Generation of cRNA probes for in situ hybridization histochemistry.

Time required

Overall=1–4 months (depending on the exposure times required for radiolabeled hybrids).

  • Creation of templates using PCR=3–4 days

  • Preparation of frozen tissue sections on poly-l-lysine coated slides=1 day

  • Generation of probes labeled with Digoxigenin-UTP=6 h

  • Generation of probes labeled with 35S-NTP=5 h

  • Hybridization=1 day (prehybridization=2–3 h; hybridization=4 h; post-hybridization=3 h)

  • 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

  • 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

  • 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

  • Create linear templates for run-off transcription of cRNA-probes using PCR.

  • Prepare sections of fresh frozen brain by cryosectioning.

  • Transcribe digoxigenin- and 35S-tagged cRNA probes.

  • Hybridize with a digoxigenin-tagged and a 35S-tagged cRNA probe simultaneously.

  • Visualize the mRNA-digoxigenin-cRNA hybrids immunohistochemically.

  • 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.

References (15)

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