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

[Frances Elizabeth Barron]

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!

Best,
~Francie

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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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