Background in murine FFPE murine tissues

From:Gayle Callis <>

If your tissue has any endogenous biotin, antigen retrieval will bring it
back into play, make sure you do avidin biotin blocking.  We routinely do
it on all tissues when dealing with SA-HRP metbods regardless of tissue.   

Some other ideas if you do A/B block and still get background. 

Put 0.05% Tween 20 in all buffers, diluents (Elias suggestion,
concentration can be increased if needed.  
Make sure the polyclonal is diluted out far enough, did you do a good panel
for proper antibody concentration??

The protein blocker can be normal serum matched to host of secondary ie
goat, 10% or so, rather than another type of protein with the mousie types
to prevent cross reactivity of species.  

Always use a secondary that is adsorbed to mouse, make sure the secondary
is not too concentrated either.  

We use DAKO AEC+ or AEC (both ready to use) for 2 - 5 min, rarely longer.  

You could be overdeveloping the chromogen, and if so, watch it develop
using a microscope with a running timer.   

'm doing IHC on FFPE nude mouse spleen, and I'm having a BIG background
issue.  I do antigen retrieval with citrate buffer, 3% peroxidase quench for
10 minutes, protein block - 30min, rabbit polyclonal antibody - 60min,
biotin goat anti-rabbit secondary - 30min, SA-HRP - 15min, AEC-12min,
Any suggestions?
Thanks a lot.

Gayle Callis
Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
Bozeman MT 59717-3610

406 994-6367
406 994-4303 (FAX)

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