RE: HELP-IHC Nonsp. Rx
Sounds like the center of the tissue is not well fixed. Is there specific
staining in the unaffected rim of tissue?
From: Greg Dobbin [mailto:dobbin@Upei.CA]
Sent: Wednesday, August 14, 2002 9:13 AM
Subject: HELP-IHC Nonsp. Rx
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.
Atlantic Veterinary College, U.P.E.I.
550 University Ave.
Canada, C1A 4P3
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