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Detection of mRNAs on Cryosections of the,,,Cardiovascular System Using DIG-Labeled RNA Probes

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The tissue samples were prewarmed to 22 C and tissue sections were prepared (4 m12 m, thicker sections may be preferred for confocal microscopy). The sections (24) were placed on silanecoated slides and immediately used or stored at 80 C (up to several months).

Prehybridization procedure

The slides were dried for 1 hour at room temperature or in an oven for 10 minutes at 50 C. The sections were treated for 10 minutes with chloroform, if the tissue was rich in lipids. The sections were fixed for 10 minutes with phosphate-buffered 4% paraformaldehyde and rinsed 3 times in 5x TE (50 mM Tris-HCl pH 8.0, 5 mM EDTA). The sections were treated with proteinase K for 10 minutes at room temperature if necessary. Whether proteinase K treatment is required, and which concentration of proteinase K is used strongly depends on the kind of tissue and the fixation. For blood vessels and myocardial tissue we used:
  • Cryosections: up to 2 g/ml
  • Paraffin-embedded sections: up to 20 g/ml
  • Methacrylate-embedded sections: up to 50 g/ml
Sections were rinsed in Tris-Glycine (100 mM Tris-HCl pH7.0, 100 mM glycine), post-fixed for 10 minutes in 4% phosphate-buffered paraformaldehyde, and rinsed in TBS (50 mM Tris-HCl pH 7.5, 150 mM NaCl) 3 times for 5 minutes. The sections were then rinsed in distilled water once (5 minutes), dehydrated in increasing concentrations of ethanol, and dried in a dust-free area. After the prehybridization procedure, the sections may be stored for a few days in a refrigerator. However, optimal results will be achieved by immediately continuing with in situ hybridization.

Hybridization procedure

Homologous probes were hybridized at 5052 C. For heterologous probes, we recommend lowe
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