[Histonet] human-on-human IHC staining

Andrea Hooper anh2006 <@t> med.cornell.edu
Wed Mar 28 07:57:19 CDT 2007

For human on human staining several options work excellently in my hands:

(1) Precomplexing primary with secondary antibody before adding to 
tissue (with appropriate blocking steps)
(2) Biotinylating your primary with a kit beforehand (not Zenon 
exactly but instead biotinylate in larger quantities)
(3) FITC labeling your primary (FluoReporter kit from InVitrogen) 
then use a rabbit anti-FITC "secondary" and an anti-rabbit polymer 
for amplification.

Steps 2 & 3 require that you have sufficient primary in an 
appropriate concentration to conjugate.

Are you sure the antibody you are working with is "good" for IHC ... 
??? These kits unfortunately won't make miracles out of antibodies 
which are not optimized for IHC. I have not tried Zenon personally 
but have heard good things. If you want, send me your Zenon based 
protocol and maybe I can help.

LMK if you need more details/information on any of the above.

>I did a quick search of the archives to look for information on the 
>use of human antibodies on human tissue, but there was not much 
>information there on this subject.  We tried the Zenon kit 
>(biotinylation) from invitrogen (as recommended by Chris van der 
>Loos), but have not had much success as of yet.  Any tips or advice 
>would be appreciated.
>Kim Merriam, MA, HT(ASCP)
>Cambridge, MA


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