Struggling with RNAscope (FISH) probe hybridization
Hi guys, I'm a first year PhD student, and have not been able to get any data for the project I got put on. My PI is new and we have only had the RNAscope protocol work once, and it was the first one they did here before I even started. My PI suggested I go to social media to ask for help. They don't want to "waste" reagants on using control probes, so we don't have any of those. But our C1 (650) (which are pictured in a screenshot because the photos didn't save right) is working now, and they said that should work as a control probe. This protocol worked fine at the last university they were at so we're assuming this is just user error? We even bought new probes and stuff and it's still not working.
I think our problem is that the probe is not hybridizing, because we are still seeing color, but it looks like autofluorescence from the blood vessels. Usually our DAPI looks good, but not in this picture here. Maybe something in the way the tech and I are preparing it? My PI has watched us prepare it and just said to be careful and not over-homogenize it because that could "kill" it.
The screenshots below show images and specifics in probe/dye. We are using the protease based V2 protocol. I just have blacked out what we are tagging (520 is highest expressor and 650 is lowest expressor). We are using 4 sections per slide, and only doing two slides at a time. At the end we add the DAPI, gold floormount, and then seal the edges with nailpolish (which has helped the tissue from degrading overnight).
The sample is 20um mouse brain that was fixed with PFA via cardiac perfusion, stored in PFA for 24hrs, stored in sucrose for 48hrs, then frozen with liquid nitrogen, stored at -80, cut at -20.
I will also be reaching our to ACDbio, but I wanted it get more imput!
