Hi Reyna,

I used quite similar protocol as yours. I think you could consider using citrate buffer before blocking the slides (0.01M pH6 at 90° for 5 min works for me). Try adding higher concentration for the primary antibody for trial purposes (1:50), but be cautious for false positive. There have been arguments whether we should incubate the sections overnight 4° or 1h room temperature, but I've tried both and achieved similar results.

You could also read the paper for reference:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6100681/

I hope this helps.

Hi Abu. First at all, thank you for your feedback.

It helped in fact to improve the signal of other antibodies, but I couldn't get Pax7+ cells. To give you some context, I was doing damage with CTX, and trying to get signal just 1day after the damage. As I was progressing in evaluating other slides, I found Pax7+ cells in those that were more than 2 days after CTX, with a potent signal at 7days after CTX! (I need to say: that I jumped of happiness the first time I observed Pax7+cells in my samples!!!). Many thanks Abu!