Hi Claire, I'm also interested in re-probing for immunostains. If you receive any advice off-list, I would be happy to hear about it.
I have an experiment that I may need to try this for, so I have done some preliminary research on the topic, but have not tried it yet. In principle, it seems reasonable to do. The only concern would be breaking the antibody-antigen interaction, without using some harsh buffer/detergent that would harm your epitope of interest. I plan to use 5 10 min washes of PBST, 0.5% -1% Tween-20 at room temperature. My reasoning is that 0.1% Tween is used routinely for washes in between antibody incubations for both Western blots and immunostains. I've mistakenly used 0.5% Tween for Western blot washing before, and it stripped the primary antibody :) If you have a membrane bound antigen, I wouldn't use detergent stripping. Either way I would confirm that your protocol works for each antibody you want to try - after stripping and washing, re-incubate with secondary antibody and see if you have any signal. This is how it is routinely done for Western blots. All of this is also presuming you are working with fluorescence detection; if you are using IHC with DAB, you will not be able to get the chromogen off the tissue even if you do remove the antibody. Invitrogen has a collection of protocols here: http://molecularprobestechnologynetwork.community.invitrogen.com/docs/DOC-1018 Let me know of your results & good luck! -Andrea Andrea Marion Graduate Student University of Illinois at Chicago [Histonet] Stripping antibodies Claire Weston cweston <@t> valasciences.com Thu Oct 27 12:30:40 CDT 2011 Previous message: [Histonet] Microm HM 560 vacutome for brain sections? Messages sorted by: [ date ] [ thread ] [ subject ] [ author ] Hi! Does anyone have experience in IHC stripping antibodies from a tissue section and re-probing with different antibodies? What is the best way to do that? If you could share your stripping protocol or experience I would appreciate it - thanks! Claire Claire Weston, PhD Research Scientist Vala Sciences, Inc. San Diego, CA 92121 _______________________________________________ Histonet mailing list [email protected] http://lists.utsouthwestern.edu/mailman/listinfo/histonet
