pcna stain problems

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From:"Jay Turner" <turnerjayd@hotmail.com> (by way of histonet)
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I am running a PCNA stain on fractured rat femora.  I am using brain as a
negative control and intestine as a positive control.  The intestine is
staining in an appropriate manner but the brain is also picking up the
primary antibody to PCNA.  The brain stain appears to be nuclear and
organized.  The positive, primary antibody is conjugated to HRP and DAB is
the chromogen.  Endogenous peroxidase activity is quenched in 3%H202 for 5
minutes prior to application of the antibody.  Also, a negative antibody
(same immunoglobulin with a non-interfering specificity conjugated to HRP)
was also used.  This produced no staining in any of the tissues (brain,
bone, intestine).  This makes me believe that it is not the procedure but
the primary antibody that is causing the problem.  I have run this stain
before on  brain with no positive staining.  Any idea what is happening?


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