I found the optimal antibody dilution and incubation time to stain cells of fish brain sections (50μm thick).
However I need to stain those cells in thicker brain sections and I was wondering what are the criteria to apply, if any, for the antibody dilution and incubation time so I can get results comparable to thinner section staining (i.e. Increasing the incubation time according to the thickness).
Looking forward to your feedback.
Hi Angelo Guadagno
I increase the incubation time for primary (15hrs) and secondary antibodies (1.5hrs) for IF in the mice brain sections (40um sections). I have observed that increasing the antibody dilution leads to increase in background staining.
Also increasing the permeabilization and blocking helps in better antibody penetration and minimises the background.
Thanks,
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