I am certain that the RNA that we are using for colorimetric ISH has not degraded. Some of the fish have started showing faint staining, but they haven't properly stained and it has been an hour since adding staining solution.
1 hour is not long at all for an in situ hybridization colorimetric assay. As long as the background is clean, you can let the color reaction go for many hours, even overnight. Sometimes for low level expressed genes, the whole mount in situ hybridizations are stained for 2 days.
1. Perform colorimetric assay at 37 degrees in dark
2. Change NBT/BCIP reaction mixture with 30 min intervals.
For some staining protocols you can a use microwave oven to reduce times. It can also improve the signal. Do a research on "using microwave oven in staining and hybridization".
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