[Histonet] RE: Anti-DIG peroxidase problem

From:"C.M. van der Loos"


   Dear Mikael,

   Reading  your  message  I  have  two  remarks  that might explain your
   strange findings:
    1. The   used   chromogens   are  of  stong  influence to  the  final
       sensitivity/efficiency of a detection system. In your case I guess
       that  with anti-DIG/AP, NBT/BCIP was used which is one of the most
       sensitive/efficient     chromogens     available.    Far    better
       than most peroxidase    chromogens.   Your   remark   you   wasn't
       successful detecting  bio-probes with peroxidase points a bit into
       this  direction.  Have  you  tried  for example TrueBlue or Vector
       NovaRed     as    peroxidase    chromogens.    Both    are    very
       sensitive/efficient!
    2. Obviously  you are not an enthusiastic kit-user (just like me...).
       What did you do in the tyramide amplification step? Especially the
       peroxide concentration should be very very low here, otherwise you
       will get and adverse effect and certainly no amplification!

   Hope this helps.

   Chris van der Loos, PhD
   Dept. of Pathology
   Academic Medical Center M2-230
   Meibergdreef 9
   NL-1105 AZ Amsterdam
   The Netherlands


   Date: Tue, 06 Dec 2005 09:08:59 +0200
   From: Mikael Niku 
   Subject: [Histonet] Anti-DIG peroxidase problem
   To: histonet@lists.utsouthwestern.edu
   Dear Histonetters,
   I'm trying to set up tyramide amplification for my in situ
   hybridizations with digoxigenin probes.
   Unfortunately it seems as if the anti-DIG-POD by Roche doesn't work as
   well as the anti-DIG-AP I'm normally using.
   Basically, I get a weaker signal with anti-DIG-POD + tyramide
   amplification than with unamplified anti-DIG-AP.
   I have also tried biotinylated probes to avoid using anti-DIG-POD
   altogether, but so far I haven't got any signal at all with them.
   Any ideas, anyone?
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