I want to do immunofluorescence on cytospun FFPE samples. The samples have been dewaxed, exposed to proteinase XXIV, then cytospun onto slides. I am trying to then stain for a nuclear protein (p63) but not having success.
Does the proteinase XXIV destroy all proteins in the cell (including the cell membrane)? Am I inadvertently destroying my p63 protein?
Yes proteinase XXIV can digest membrane proteins. But I am not sure weather you are carrying out your immunoflorescence under permeabilized or non-permeabilized conditions. If you are carrying out under nonpermealized condition, I dont think the proteinase could digest nuclear p63 protein until and unless the proteinase affect the integrity of cell membrane. You can check dapi staining in the presence of proteinase under nonpermeable condition. If you find diffuse type of staining then, I am sure proteinase affect membrane integrity thereby can digest any protein in it ways.
Hi. Thanks for answering. So I guess the membrane is already pretty permeable as its FFPE tissue but after the proteinase XXIV (once it has been cytospun onto the slide) also use PBS-Tween detergent to permeabilise. The slides also undergo citrate ph 6 antigen retrieval again once the proteinase XXIV has already been used. I guess one question I have is what's the point of the proteinase XXIV? If its only role is to permeabilise the membrane, then can I leave it out all together if I am doing citrate antigen retrieval anyway with PBS -Tween?
My DAPI stain is not diffuse and stains the nuclei very well which I suppose indicates that the membrane is permeable?
Yes you are right, as your purpose is immunolocalization of p63 protein, I have not encounter any procedure for using proteinase XXIV. And I am 100% sure that you can leave it out.
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