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

r temperatures. The hybridization solution (50% formamide, 2x SSPE buffer, 10mM DTT, 1mg/ml herring sperm DNA, 500 g/ml yeast tRNA, 1 mg/ml BSA) was denatured for 10 minutes at 80 C. The sections were preincubated in a humidified chamber for 2 hours in hybridization solution. The prehybridization solution was removed, and hybridization solution was added (concentration of the DIG-labeled RNA probe: 0.3 1 g/ml). The tightly sealed chambers were incubated overnight in a shaking water bath at 50 C.

Posthybridization procedure

The hybridization solution was removed by thoroughly rinsing the slides in 4x SSC. The slides were washed twice in 2x SSC for 15 minutes at 50 C and twice in 1x SSC for 15 minutes at 50 C. To remove nonspecifically bound DIG-labeled RNA probes, the slides were incubated with RNase A (10 g/ml NTE: 500 mM NaCl, 10mM Tris-HCl pH 8.0, 1mM EDTA) for 10 minutes at 37 C. The slides were then washed twice in 0.1x SSC for 10 minutes at 50 C.

Detection procedure

For detection, the DIG Nucleic Acid Detection Kit was used. The sections were washed for 5 minutes in buffer 1. Nonspecific background was blocked by incubating the sections for 1 hour in buffer 2 (buffer 1 with 0.5% Blocking Reagent).

  • Alkaline phosphatase conjugated antibodies

The sections were incubated with anti-DIG antibody conjugated with alkaline phosphatase (Fab-fragments) (dilute 1:5001:1,000 in buffer 2) for 1 hour at room temperature, then rinsed thoroughly in buffer 1 containing 0.05% Tween, washed twice for 15 minutes, and incubated for another 15 minutes in buffer 3. The slides were then incubated with an appropriate amount of staining solution in a humidified chamber for 30 minutes to 24 hours (stainin
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