RE: IHC problem: secondary reaction against sheep

From:"Johnson, Teri"


It's possible to just plug in a biotinylated anti-sheep secondary antibody to a kit which uses a streptavidin/enzyme (peroxidase or alk phos).  Buy the secondary, try different dilutions on your tissue to find the best signal with the least background.  If the manufacturer recommends a dilution range of, for instance 1:500-1:5000, start with 1:250 and try that, and also 1:500, 1:1000, and 1:2000.  If your slides don't show good staining, then try other dilutions.  Too intense, extend the dilutions.  Too weak, try again at 1:25, 1:50, 1:100, etc.

You can also try an anti-sheep that's directly conjugated with peroxidase or alk phos.  It's less sensitive than biotin-streptavidin systems, but you won't have to worry about blocking for endogenous biotin that way.

It's not that difficult to move away from kits either, if you want to go that route.  I recommend you getting the Immunohistochemistry staining manual from Dako Corp.  That should give you some good basic information.  

You never said what species you're testing.

Good luck,

Teri Johnson

-----Original Message-----
From: []
Sent:	Thu 9/26/2002 3:43 AM
Subject:	IHC problem: secondary reaction against sheep

Dear histonetters,

I need your help again. I'm not experienced in IHC. 

Unfortunately, I must to test an antibody against pepsinogen type II from 
My problem regards the secondary reaction: as far as I know, there aren't 
kits to develop the secondary reaction against an antibody from sheep and 
it could be become complicated to set up every single step of the 
secondary reaction. 

What shall I do? Does anybody know if there is a kit to resolve this 

Thanks in advance

<< Previous Message | Next Message >>