Hi @achamess, thanks for all your helpful input on this forum. I’m trying to do RNAscope in a tdTomato mouse line - could you advise on how to recover the fluorescence with immunolabelling after the RNAscope procedure?
Thanks!
https://rockland-inc.com/store/Antibodies-to-GFP-and-Antibodies-to-RFP-600-401-379-O4L_24299.aspx
1:500 after RNAscope. @MGM has had success with this.
Good luck.
1 Like
Hi! sorry for the delayed response! We got it working really well. We are doing rats DRGs that I’m fixing on 4%PFA for 16 hours at 4C (we have not tried a shorter fi, and then 10% sucrose for 1h, 20% sucrose for 1h and finally 30% sucrose for 24 h and to storage. We are slicing at 12 microns and then applying the fresh frozen protocol (minus the slide fixation as they are already fix). We are using the TSA system at a 1:3000 dilution. We have tried the CGRP probe (CALCA) and 4 different custom made probes and have positve results with all of them. We are also working on perfecting a protocol with Nissl staining instead of the DAPI, to only get neuronal staining.
Awesome! Thank you for sharing. Nissl sometimes works after RNAscope but it’s not as nice as untreated. I think maybe the protease messes up the ribosomes, which is what Nissl binds to. Keep us posted. Share a pretty picture if you’re willing. @MGM might be able to comment on Nissl.
Beautiful! Thank you. Great signal. Is that the Opal 520 or FITC? See the comment above about Opal. I think it works much better.
the second one is CGRP which is very high express
we did FITC and so far we are happy with the signal. We may try the Opal as well.
Amazing! Yes some of them are so strong they fill up the whole cell. The RNAscope ‘positive’ control has never been great. It’s there, but always weak. Same in spinal cord. I prefer to establish my own positive control (CGRP now is a good one for the future) that you know should be there.
Yes, that is why we decided to use CGRP as an internal control of something we were certain was express in the DRG! I’ll upload more pics as we get more results!
Fantastic! I’m so pleased about your results and I’m very grateful that you’re willing to share your experimental details and results. No need to reinvent the wheel. We researchers waste so much time doing what others have already figured out. Science moves much faster when people like you share their experiences. Good luck with your ongoing experiments.
If he decided to change the opal 520 instead of the FICT, can you combine with the TSA CY3 and Cy5 or we will need to switch completely to the Opal?
You can combine with the Cy3 and Cy5. This is what we do.
Have you tried the other Opal dye equivalents, 570 and 690? Any better or worse than Cy3/5? We’re about to try out the new v2 kits but havent got the fluorphores yet.
@gcorder I haven’t. The Cy3 and Cy5 work well enough. But if you just want to get the Opal kit, that might not be a bad idea so you don’t need to get the FITC, which I don’t think this very good.
Hey
I’m using fixed frozen tissue, and lately I’ve noticed the nuclei blebbling.
Have you encountered this issue before?
My tissue prep includes the following: fix OVN 4%PFA, 10/20/30% sucrose, oct, and frozen on dry ice. Then fixed frozen protocol, was 1XDPBS>Pretreatment 10min H202, 5min Retrieval Reagent and 30min Protease III.
Thanks so much!
Can you show some images of the blebbing? And have you done RNAscope before without blebbing?
Hello,
Some questions:
Do you skip the boiling (Target retrieval) with the v2 kit?
Do you do the protease step in v2 for 30 min?
My tissues have fallen off (I use super frost plus slides)…My slides were stored in -80C, and were from frozen fixed tissue (overnight 4% PFA, 30% sucrose overnight), and I followed the frozen fixed tissue protocol. Do you have any idea why they fell off? Sadly, I added my probes, etc., and didn’t know they had fallen off until I reached the confocal microscope.
Thank you!
Hi.
What tissue are you using? If DRG or spinal cord, yes I skip the boiling. I use the fresh frozen protocol even though my tissues are fixed. Overnight fix is too much. I’d do 2-4 hours.
I use protease for 30 mins at RT, per the fresh frozen protocol.
Did you let your tissues dry on the slide at RT for at least 15 mins? That helps. See comments above. You might also use new slides. Maybe you got a weird batch of super frost.
Good luck