Hello all,
I have been instructed to perform dual-labeled TUNEL + IHC staining on paraffin sections by the boss. Just wanted to know if any out there have any experience or advice while I construct my protocol.
We are using the Promega DeadEnd Fluorometric system. My first question regards the protocol from Promega. It states that you need to perform two 4% PFA fixation steps after hydration (one on either side of the permeabilization step). What is the point of this since the tissue has already been fixed in PFA originally? This would make sense for frozen sections but it is under the Paraffin section protocol. Since I have to perform antigen retrieval for my primary antibody I am thinking this should be done after all fixation steps, does that seem reasonable?
I tried just the TUNEL (with antigen retrieval) using either proteinase K or 0.2% Triton-X permeabilization to see if I could bypass the PK step (since I do not want to chew up my epitopes which lie within melanosomes) but it didn't work with Triton-X alone. Second question: Will proteinase K attack antibodies? I am performing the TUNEL first as it seems the antibody-antigen complex makes a better target than just the epitope.
Final question: what is the role of using 0.85% NaCl at the end of the TUNEL hydration steps? Just wondering as I don't typically do this for IHC.
Much appreciated.
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