I have been doing immunostaining with frozen skeletal muscle cross sections for quite some time and I have routinely counterstained with a nuclear marker (hoechst or DAPI) with no problems. Recently, all of the nuclei in the sections appear like they have exploded or are leaky and I have no idea why. It appears that the problem is sometimes attenuated when I fix the sections prior to staining, but almost all of my antibodies do not work well on fixed tissue. This problem can be seen with both DNA markers and nuclear protein antibodies. Does anybody have any idea why this is happening and/or how I can fix it? Thanks ahead of time.
Maybe it is the thawing process that damages the nuclei. In this case you can try Paraffin embedded IHC.
Can you show us a picture? It sometimes happens when you permeabilize with Triton-X, at any step during the protocol. Is this the case?
I thought it was but I cut out any detergent and it still persists. I'll take a picture and upload it.
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