HELP-IHC Nonsp. Rx

From:Greg Dobbin

Hello All,
My problem seems to be definitely an artifact due to a technical 
problem, but I can't figure out what!?

I am getting a strong nonspecific reaction at the centre of all my test 
sections (like a small brown fingerprint), while my deletion controls 
(one for each test) are completely negative. 

What could be causing this? I would think a problem with washing 
would be more generalized, and a problem of drying would be the 
reverse, nonspec. rx on the perimeter of each section.

I am staining formalin fixed parraffin embedded tissues (5 um). I 
quench with 3% H2O2, block with Dako Universal Blocker, incubate 
the primary Ab overnight at 4 C, block for biotin with Dako Biotin 
Blocking System, label with Dako LSAB Steptavidin kit and DAB, 
and counterstain with hematoxylin. The wash buffer is PBS (pH 7.4) 
with 0.3% triton, 0.2% saponin, and 1% bovine serum albumin. The 
wash buffer was used to dilute the primary antibody.
Greg Dobbin
Pathology Lab
Atlantic Veterinary College, U.P.E.I.
550 University Ave.
Charlottetown, P.E.I.
Canada,  C1A 4P3
Phone: (902)566-0744
Fax: (902)566-0851

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