Hi everyone!
We have formalin fixed paraffin embedded thyroid carcinoma tissues collected over past few years. Now I want to check expression of few phosphorylated kinase proteins on those tissues by immunohistochemical staining.
Can anyone suggest, with protocol, if it is possible to check expression of phosphorylated protein on formalin fixed paraffin embedded tissue, or do I need to follow different fixation procedures for that purpose?
Thank you very much.
Hi,
It depends on which antibody you want to use. In the data sheet you should find the best protocol. Of course you should validate the protocol and, using positive and negative controls, adapt it to the tissues (and the fixation) you have.
It depends largely on how stable is the phosphorylation status of your proteins of interest. It also depends on the size, thickness and density of the tissue, especially if you did fixation by immersion rather than perfusion, and whether the tissue was fixed immediately after sampling. If the phosphorylation lasts long enough that the formalin had time to penetrate, you should be able to get a good signal. If the phosphorylation is labile, and your tissue samples were solid and thick, you might get positive staining only at the edges of the tissue, and nothing in the center, because the phosphorylation in the center was lost before formalin had time to penetrate there. Of course, there also needs to be reliable antibodies for the phosphorylation sites of interest, which are known to work in formalin-fixed tissue. A good antibody supplier should be able to provide data on whether their antibodies work in formalin-fixed tissue, and offer a garantee if they don't.
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