Dear J, I agree with Rene Busa on his assessment that thawing and refreezing is very very bad for your epitopes. You don't do it with antibodies, you don't want to do it with your antigens either. Keep the slides you will not need frozen, taking out only what will be needed. Work quickly as exposure to the air will start the condensation process.
I also agree that there does not need to be an ethanol rehydration step. That might be useful as a permeabilization step, but you only need to hydrate your tissue with buffer. You might not have realized that alcohol can permeabilize cells (even though they are already exposed through sectioning) and also affect the protein folding, so if your antibodies are already working using this scheme you might see a difference if you quit doing it. You also might not, so it could be useful to test it. As to whether to use heat to thaw, you can try putting the slides in front of fans at room temperature, or you can try fan forced ovens set at 25 or even 37 degrees if you are worried about heat. I have worked with a researcher who used unfixed cryosections of brain and put them in a 95 degree oven for 2 minutes prior to freezer storage. They were still able to get antibody and mRNA staining from their sample. I have also read accounts of people using a hair dryer (blow dryer) on the heat setting on them as well with no ill effect on their published studies. Who is to say they might have had to try multiple antibodies to find one that would work under those conditions? The only way to know if heat or other conditions will negatively affect your target protein is to test it empirically. Teri Johnson, HT(ASCP)QIHC GNF Histology Lab Manager Genomics Institute of the Novartis Research Foundation 858-332-4752 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
