[Histonet] RE: Red chromogens

From:"C.M. van der Loos"

Dear Rena,
Too much levamisole cannot result in no staining. If you have used one drop (from the Dako concentrate??) per ml of substrate that should be OK (0.5 mM levamisole is considered as optimal). Too much of levamisole may result in somewhat weaker staining ultimately as levamisole inhibits the activity of all AP isoenzymes except the one from intestinal tissue. Your AP label is isolated from calf intestinal tissue. 
I can strongly recommend you to test the new Dako Permanent Red substrate (if already on the market).
One suggestion: if you are a PBS user the phosphate ions from that buffer inhibit AP activity dramatically. Change to TBS or insert 2-3 washing steps with a non-phosphate containing buffer between your final PBS washing step and AP visualization.

Hope this helps.

Chris van der Loos, PhD
Dept. of Pathology
Academic Medical Center
Amsterdam - The Netherlands

----- Original Message ----- 
>From  Rena  
Date  Wed, 29 Sep 2004 21:30:04 -0400 
To  histonet@lists.utsouthwestern.edu 
Subject  [Histonet] Red chromogens 
I have been having some trouble with some Abs stained with New Fuchsin
and permanent red. Recently I was asked if I were using Levamisole in
the permanent red and if so how much? I was told too much Levamisole in
permanent red would result in no staining. I have used 1 drop per ml for
both New Fuchsin and permanent red, which is the appropriate amount.  We
have run both DABS and either permanent  red or New Fuchsin with the
same AB from the same vial with vastly different results. Any ideas?
Rena Fail  

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