Hi all :)
I am trying to perform smFISH followed by immunostaining on drosophila testes but when I check how the staining looks I have no signal from the smFISH probes (while the antibody is fine). I tried to invert the 2 staining and perform IF first and smFISH after. This improved the smFISH but somehow worsen the IF.
In the past I have worked with Drosophila embryos and I never has any issue in performing smFISH followed by IF, could it be a tissue specific problem? Which approach would you suggest to try?
Thanks :)
(smFISH and IF alone work both perfectly fine on testes)
I encountered similar problem with some tissue types. My suggestion will be- First incubate your samples with Primary antibody then crosslink antibody with proteins using 4% paraformaldehyde for 30 min. After washing with PBS proceed with smFISH. After FISH, incubate with secondary antibody.
Thank you Rahul. This is interesting, so you would fox again after the primary. Have you ever tried to do primary, then secondary then fix and then smFISH? Or do smFISH, then fix and then primary and secondary?
As per my experience, fixing after secondary antibody incubation or smFISH destroys the signal. Better to fix it again after primary, this is what i am doing routinely.
Rahul Kumar thabk you very much for your suggestions. I will give it a try and let you know!
It seems to me that trying different rounds of fixing (after the primary antibody) might help to stabilize the interactions therefore improving the quality of your experiment after incubation with secondary antibody.
On the other hand, using primary antibodies (nanobodies also) chemically labeled with fluorophores algo might help As you don’t need to use secondary antibodies, eliminating un cesar washing/incubation steps.
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