Becky,

I would do 3 controls if you have not already done so, secondary only,
avidin-HRP only and detection only. this would tell you where the problem
lies. I sometimes dilute my avidin-HRP and am able to maintain sensitivity
and decrease or eliminate background staining. Problem may even be with your
negative serum controls.
Good Luck,

Cynthia


-----Original Message-----
From: becky riesen [mailto:beckyr@naples.net]
Sent: Friday, August 01, 2003 9:48 AM
To: histonet@pathology.swmed.edu
Subject: more info


I'm sorry about not giving enough information.  I have had several 
responses asking for more details.  So, I'll try again!

I am performing IHC staining.  I am using a DAKO autostainer with 
the LSAB detection system.  I use DAB for my chromagen.  My negative 
mouse and negative rabbit serum slides have quite specific staining 
in the granlocytic series of WBC's(No matter what pretreatment - 
I apply no pretreatment, Proteinase K, or use Microwave at pH 6 and 
10).  Microwave pretreatment is the worst, but I get the staining 
most of the time.  Polysegmented neutrophils seem to be the main 
culprit.  Most of the times my Docs can read through this, but they 
are requesting my experimentation to find a "cure" for the situation,
so they can honestly say, "no staining was seen in the negative 
control". One of the suggestions I have received is to do an Avidin/Biotin 
Block. I will give this a try, but if there are any other ideas out 
there:  DON'T hold back!  THANKS AGAIN! 



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