AE1/3 Cam 5.2 cocktail -Reply
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From: | Richard Cartun <Rcartun@harthosp.org> |
To: | lhadley@iusd.iupui.edu, histonet@pathology.swmed.edu |
Reply-To: | |
Content-Type: | text/plain; charset=US-ASCII |
We have been using a cocktail of CK-CAM5.2 and CK-AE1&AE3 for years. As usual, the optimal concentrations of the components will depend on your fixation/processing, antibody incubation time, detection system, chromogen, etc. My experience with making antibody cocktails is as follows: titrate each antibody in the cocktail to its optimal concentration then, when preparing the cocktail, use 50% (half) of what you would normally use. I have found it necessary to do this because when antibodies are used in combination there is an "amplification" that occurs that results in "over-stained" slides in most cases.
For example:
If you use CK-CAM5.2 at 1:100 and CK-AE1&AE3 at 1:1,000, you would prepare the cocktail so that the CAM5.2 dilution is 1:200 and the AE1&AE3 dilution is 1:2000. We dilute our primary antibodies in PBS with 1.0 % BSA and 0.1% sodium azide. Obviously, some "fine-tuning" of the concentrations may be needed.
R. Cartun
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