Hello all,
I am having trouble with antibody penetration of Iba1 staining in my tissue, i can only seem to get about a 30-35 z-stack. Additionally I have a really hard time mounting this tissue, it seems like either i let it over dry and the PBS creates salt stains on the tissue or I don't let it dry enough and when I try to mount the tissue folds over itself when.
Any suggestions would be appreciated!
Hi Malik, have you looked at fixing your tissue via the use of a 15second microwave? It's an old method, but it has worked for some people in the past. The difficulty in 'the folding of the tissue'? could be as simple as static electricity. I am statically charged and I have seen samples lift turn and fold due to static in the glassware, I usually spray crospray gentl onto glassware before I use it. You might try mounting in a cold room4C or in a cryostat at -16--20C? Just a few ideas. There is a jounal called Methods in Histology available from many years ago, but the old methods still work.
I agree with the above answers but would also recommend washing your slides in ddH2O after staining is complete and before mounting to reduce salt crystallization.
https://www.nature.com/articles/s41598-019-44897-7.pdf?draft=marketing
https://www.thermofisher.com/nz/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/ihc-troubleshooting-guide.html#2
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