[Histonet] RE: recommended ICC protocol for use of Conjugated mouse antibodies with unconjugated mouse antibodies

Frances Elizabeth Barron fbarron <@t> stanford.edu
Wed Nov 7 13:43:30 CST 2012

Hi All,

Hope this day finds you well.

I have two antibodies in the lab that I would like to use together for ICC. One is a mouse anti-human pre-conjugated antibody, the other is a mouse anti-human unconjugated antibody. My question is....how to get these guys to play nice with one another or should I not be worrying about it? 

My thought is that I have to do this sequentially, (stain first with the unconjugated, stain with the secondary, then stain with the pre-conjugated antibody?). However, I'm questioning whether or not this is necessary. Would the mouse secondary react to a conjugated antibody? Does the process of conjugating hide the secondary epitope?

I have had bad luck with loosing substantial signal from the first round of primary/secondary antibody staining in a sequential staining, so I'm loathe to do it (appreciate any advice on that issue if you have it!). I can find one of the antibodies raised in another animal, but it isn't validated in human or ICC (just predicted to work). Just trying to see if I can use what I have before I go out and buy another antibody that may or may not work.

Appreciate any help with this!



Francie Barron, Ph.D.
Postdoctoral Fellow, Joseph Wu Lab

Stanford University School of Medicine
Lorry I. Lokey Stem Cell Research Building
265 Campus Drive, Room G1105
Stanford, CA 94305-5454

Phone: (650) 724-5564 or (650) 724-9240
Fax: (650) 723-6272


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