Hi:
I am performing immunofluorescence staining on 100 um 4%PFA fixed pancreas sections using our self-made polyclonal primary antibody. Tissue was gradient dehydrated in methanol diluted in 0.2% NP40. The sections were blocked with 1% BSA, 4% FBS, and 0.1% Tween-20 in PBS for 1 hr. But end up with a poor staining result under confocal examination. Is there any possible solution? Thanks!
Were the isolated single cells fixed and dehydrated like the sections were?
It depends on what you mean by "poor" labeling. Antibodies are large molecules that do not penetrate so well in tissue. I once determined with confocal imaging that one or our antibodies used on glutaraldehyde-fixed brain tissue sections had penetrated only 8 µm on each side of free-floating 40 µm sections. So if you use 100 µm sections, it could just be that the antibody did not reach the "inside" of the sections but only the surface, which will show under confocal imaging on optical sections taken from the middle of your 100 µm-thick section.
Also, on tissue sections, your antibody could cross-react with other molecules present in the tissue that are not present when isolating the cells.
Again, it depends on what you mean by poor labeling.
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