Hi @achamess, I’m new to RNAscope and am interested in trying your methods for DRGs! I had a couple of questions on details…
- Do you perfuse with saline or PBS prior to PFA, and does the rate of perfusion matter?
- Could I substitute PFA with NBF? (There’s a giant jug in the lab but I heard it can go bad over time, even with the methanol…?)
- For sample pretreatment, do you skip the hydrogen peroxide step prior to protease IV when using the v2 kit?
- It seems like the fluorescein TSA didn’t work out well for you earlier… would you then recommend Cy3 and Cy5 if co-staining? How would you decide which gene to put in which channel?
- Is there anything to be particularly careful of in terms of avoiding RNases and RNA degradation during any of the steps?
- Finally, have you tried doing RNAscope in transgenic lines that express endogenous fluorescent proteins? We have a tdTomato line I’d love to use but I’m not sure if the protein will survive the RNAscope procedure.
I don’t have much experience with ISH or microscopy in general so any tips and advice would be much appreciated! Thanks so much for your help!