liver/IHC/background staining

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From:Gayle Callis <>
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You may have more than endogenous peroxidase giving you fits and at the
concentrations you are blocking with (using H2O2), bet that is gone.  You
did not say if you were blocking for endogenous biotin?  Liver has a high
level of this, and antigen retrieval is known recover endogenous biotin
subdued by NBF followed by processing in paraffin. 

Use an avidin/biotin blocking kit per vendor instructions, DAKO, Vector,
etc all have excellent products, block AFTER retrieval. 
A good test is to take one slide and block with avidin/biotin kit, and not
block the other, if it is gone, voila!  you win!    

Other ways to fight this problem, some protein super blockers containing
casein (casein blocks biotin) or some simply add 5% casein to buffers,
diluents, etc during staining.  That concentration could/may vary, more
suggestions here???    Some people have used nonfat dry milk from grocery
store, or buy casein from Sigma, etc.  I don't use casein, someone else
could give better details here.   

If background is still sticking around, another tweak or three may be
needed to fine tune annoying background away, not caused by endogenous
perox or biotin.

Good luck

Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303

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