Hello everyone! I am trying to stain mature neurons in culture for pre and post synaptic markers. I used to fix my cells 20 min at 37C with 4% PFA. Synaptophysin looks amazing unfortunately I couldn't achieve PSD95 puncta. I read that the best fixation strategy might be a short PFA fixation or an ice-cold methanol fixation. As I have several samples already fixed, does someone know if a post-fixation incubation with methanol could help me visualize the PSD 95 puncta? or does someone have the ultimate protocol? also with the antibody cat number?
Best
Telma
Well, try fixing with the good old buffered formalin. Formalin is better than PFA. Sounds strange but three facts makes it a better choice for immunocytochemistry: 1) Formalin contain 10-15% methanol. This should help in fixing. 2) Its much faster acting, and 3) no need for antigen retrieval.
You might also take a second look at your primary antibody. Usually, if immuno does not work, it means problem with the primary antibody. Does your PSD95 antibody work in western blotting in your sample? Try a different antibody and try also with 1% triton-x extraction.
Regards
Hi Telma,
I have the same problem, can you let me know if post-fixation incubation with methanol helps visualize the PSD 95 puncta?
Thanks
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