Dear collegues,
how do you would perform Immunofluorescence staining on thick brain slices (around 400 micrometres) upon acute slices pharmacological manipulation or electrophysiology?
-cut and then stain or whole mount stain and then cut?
Do you have references (written/videos) for it?
Thank you
Viviana Triaca
Hi there,
There is really few papers in which what I am going to say is clearly stated but:
You don't need to cut.
I have done this experiment with 350um slices
(Article Melanopsin for precise optogenetic activation of astrocyte-n...
)[supplementary figure 2, protocol in material and methods]
and I can tell you that if the antibodies you are using are good enough you can get high quality images without cutting. But it is really important to have good antibodies. You will probably need to adjust (increase) incubation times with antibodies, washes, and also the concentration of detergent.
Check also this paper, in which they perform IHF on organotypical slices without cutting
https://pubmed.ncbi.nlm.nih.gov/17406491/
However, if you need to cut the slices or just want to do so, cut first and then proceed with the IHF protocol.
Hope this helps,
Cheers,
J
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