ProtocolHighly sensitive radioactive in situ hybridization using full length hydrolyzed riboprobes to detect α2 adrenoceptor subtype mRNAs in adult and developing rat brain
Section snippets
Type of research
- 1.
Detection of low abundance mRNA expression in developing and adult brain [13].
- 2.
Quantification of mRNA expression in developing and adult brain [16].
- 3.
Simultaneous detection of mRNA expression and receptor binding sites in adjacent sections 15, 16.
- 4.
Simultaneous detection of mRNA expression and acetylcholinesterase (AChE) activity in adjacent sections [1].
Time required
The entire procedure runs over several weeks. From the time when the tissue was first removed from the skull to analyzing the sections through darkfield microscopy takes two to eight weeks, depending on the expression intensity of the target mRNA. However, the protocol was subdivided into several parts and the time required for each step depends on the number of slides processed. The times given in here were estimated for 25 slides:
- •
Tissue fixation, 120 min.
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Probe preparation, 180 min.
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Hydrolysis,
Material
Animals: Adult (60 days or older) Sprague–Dawley rats and postnatal rats (Harlan, San Diego).
Slide preparation
- 1.
Place glass slides in a metal rack and wash overnight in diluted detergent (liquinox) solution.
- 2.
The next day rinse the slides in hot running water for 15 min, followed by a final wash in distilled water.
- 3.
Place the slides in 10% HCl/90% ethanol for 20 min, rinse thoroughly in running distilled water for 15 min.
- 4.
Briefly dip slides in gelatin solution (1% w/v gelatin, 0.05% w/v chromium potassium sulfate) and allow to drain for 30 min.
- 5.
Dry the slides at room temperature (RT) in a dust-free environment
Specificity of the probes
The specificity of the probes was tested by comparing riboprobes synthesized in the sense and antisense orientation in adjacent sections. A good sense probe should have low levels of non-specific hybridization and no specific hybridization pattern of its own, as seen for the long α2A and α2B and short α2A(i3,4) and α2C(i3) sense probes (Fig. 1A,A′,B,C′). However, the sense riboprobes for α2B(i3) and α2C did exhibit non-specific hybridization in cell dense layers of the hippocampus (Fig. 1B′,C).
Discussion
In situ hybridization is a powerful tool allowing the detection and quantification of specific mRNAs expressed on a single cell level. We developed a highly sensitive and selective method to detect low levels of mRNA expression in fresh fixed tissue, which allows the simultaneous detection of functional proteins from the same brain.
Slide preparation
- 1.
Place glass slides in a metal rack and wash overnight in diluted detergent (Liquinox) solution.
- 2.
Rinse slides in hot running water for 15 min, followed by a final wash in distilled water.
- 3.
Place slides in 10% HCl/90% ethanol for 20 min.
- 4.
Rinse slides thoroughly in running distilled water for 15 min,
- 5.
Briefly dip slides in gelatin solution (1% w/v gelatin, 0.05% w/v chromium potassium sulfate) and allowed to drain for 30 min.
- 6.
Dry slides in a dust-free environment at RT overnight
- 7.
Incubate slides in a fresh
Essential literature references
Refs. 2, 6, 10, 11, 12, 17.
Acknowledgements
This work was supported by PHS grant NS 30109. The authors thank Dr. K. Lynch for providing the α2 adrenoceptor cDNAs. We also like to thank Dr. Jan Smith, Trez Brown, Kathy Gallardo, and Dee Stoveken for their technical assistance and valued discussions in optimizing the method.
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2012, NeuroscienceCitation Excerpt :The newly generated antisense probes for NKCC1 and KCC2 exhibited the expected hybridization pattern (Plotkin et al., 1997; Wang et al., 2002; Stein et al., 2004), and the sense probes showed very low or no hybridization signal indicating negligible non-specific hybridization (Fig. 1) and suggesting specificity for the NKCC1 and KCC2 templates. In situ hybridization was performed as previously described (Winzer-Serhan et al., 1999). Briefly, slides containing coronal brain slices through the hippocampus were pretreated for 10 min with protease K (0.1 μg/mL), acetylated, dehydrated using increasing concentrations of ethanol (50%, 70%, 95%, and 100%) and dried in a cold air stream.