[Histonet] Stripping antibodies

[Andrea Marion]

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
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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