[Histonet] RE: Anti-DIG peroxidase problem
C.M. van der Loos
c.m.vanderloos <@t> amc.uva.nl
Tue Dec 6 13:15:01 CST 2005
Reading your message I have two remarks that might explain your
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
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
NL-1105 AZ Amsterdam
Date: Tue, 06 Dec 2005 09:08:59 +0200
From: Mikael Niku <mikael.niku <@t> helsinki.fi>
Subject: [Histonet] Anti-DIG peroxidase problem
To: histonet <@t> lists.utsouthwestern.edu
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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