This is my first post on this website. I hope you can help my with a problem.
We was established the optimal conditions for the immunohistochemistry using fluorescence against a transcription factor in paraffin embedded tissue. Suddenly, without any changes in the protocol, the staining didn't works properly, we can't detect any staining often and we we have staining, it is dirty and weak. We are using microwave-mediated antigen retrieval in citrate buffer and HRP-conjugated secondary antibody with amplification using TSA system. Tissue is paraformaldehyde fixed and paraffin embedded. We have already change all the buffers, the primary and secondary antibodies, the TSA system, we check different tissues positives for our protein and we are success randomly. Some months ago the staining worked in the 99% of the cases. Maybe some of you have had a similar problem and could help me? I ran out without ideas that what could be happening!!
Thanks in advance!