I need to make immunohistochemistry on rat tissue (spleen and skin). The tissues were fixed 24 hours in formol and 4 weeks in ethanol. I can use cytokeratin and CD68 on these tissues but I am still blocked with CD45 and CD3.
I tested many antigen retrieval :
Heat antigen retrival : citrate pH6, pH7, EDTA pH8, Tris EDTA pH9,
enzyme : trypsine, pronase, proteinase K : with ant without citrate pH 6
And the results are still negative with all the tests. Does anyone have an idea?
Better to check first for Ab recognizing fixation resistant epitopes.
A few anti CD3 clones or ani CD45 are claimed to work on formalin fixed tissues:
http://www.nordiqc.org/Epitopes/CD3/CD3.htm
http://www.ihcworld.com/_protocols/antibody_protocols/cd45_pharmingen.htm
https://www.thermofisher.com/antibody/product/CD45-PTPRC-Antibody-clone-PD7-26-16-2B11-Monoclonal/MA5-13197
Extensive water washing of at least one week might also works, without any morphology degradation BTW.
Good luck !!
J.
The problem may lay more in the ethanol fixation than in NBF. I doubt, if antigenretrieval can solve it. Maybe alternative antigens have a chance (CD5).
Gudrun is right. I overlooked alcohol for weeks in your fixation. You could try this one. Or seek for another T cell antigen.
J.
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