Are you using a manual or automated system? Bubbles under the coverslip 
soetimes "trap" reagents preventing thorough washing and hence NS staining.
Louise


>From: Greg Dobbin 
>To: histonet@pathology.swmed.edu
>Subject: HELP-IHC Nonsp. Rx
>Date: Wed, 14 Aug 2002 10:13:15 -0300 (ADT)
>
>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
>~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
>"A farmer is a person outstanding in their field."




Louise Renton
Bone Research Unit
MRC
Johannesburg
South Africa
Tel & fax +27 11 717 2298
"Time flies like an arrow, fruit flies like a banana"


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