I have been working to troubleshoot my Iba1 stain for microglia for the past few months with no avail. Looking to stain Iba1+ microglia in fresh frozen, OCT-embedded rhesus macaque brain, slices 10 um thick on microscope slides.
Conditions already used:
Fixation solution of 63% PIPES buffer + 37% PFA (4% stock) OR fix at 4% PFA alone
Fix for 10 minutes at RT, block with Donkey block (10% NDS + 0.1% Triton + 0.01% NaN3)
Primary antibody incubation
Dilutions used: 1:100, 1:250, 1:500, all attempted both 1 hour at room temp or 4 degrees overnight
I utilize the same protocol for all of my other antibodies and haven't had an issue, but this one just keeps escaping me. Does anyone have any pointers for fresh frozen macaque brains? Most of the literature I find on Iba1 staining is for FFPE or previously fixed brain tissue. I have tried a few different antibodies from different manufacturers (Novus, Sigma), but none have been remarkably successful at giving me the level of staining I am expecting to have.
Thank you!
Dear Michelle Ash,
You can try antigen retrieval with citrate buffer (pH6 or 9), either 97 deg C for 5 or 10 min or 60 deg C for 5-8h. Additionally, check the articles that cited the antibody that you are using to see the conditions that they have used.
PS. you can try first, if don't work, try the other things
Good luck.
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