[Histonet] RE: Nova Red/VIP substrate

C.M. van der Loos c.m.vanderloos <@t> amc.uva.nl
Fri Jul 8 02:20:57 CDT 2005

   Dear Louise,

   When  it comes to double staining, Vector Nova Red chromogen would not
   be  my  choice  for combining with DAB. Nova Red is not like AEC, it's
   more brownish-orange and doesn't contrast that well with standard DAB.
   You may try to shift the brown-yellow color of DAB reaction product to
   more blueish, by adding nickel- or chromium-salts.

   The  combination of standard DAB with VIP gives a better contrast than
   Nova red.

   Both  these  chromogen  kits are fully monkey-proof, don't worry about

   If   you  perform  this  sequential  double  staining  (with  two  HRP
   activities involved), please keep in mind the following:
     * it's  not  going to work for the observation of co-localization by
       mixed-colors,   just   for  two  different  cell  populations  (or
       different cellular compartments) only.
     * it's  obligatory  to apply  DAB  reaction  product  in  the  first
       staining   sequence!  DAB  reaction  product  is  the  only  known
       chromogen  that  effectively "cover"  immunoreagents  used  in the
       first  staining  sequence.  This  sheltering  effect  is  the  key
       mechanism  for  preventing  unwanted cross-reactions with reagents
       used in the second staining sequence.
     * sequential double staining works satisfactory if the first primary
       (and   secondary)   antibody   is   well  titrated. If  the  first
       antibody (or  secondary)  is used too concentrated, the sheltering
       effect from the DAB reaction product will not be effective.

   I do hope I didn't scare you off performing double staining!

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

   phone:  +31 20 5665631
   fax:    +31 20 6960389

   Date: Thu, 7 Jul 2005 15:45:59 +0200
   From: louise renton <louise.renton <@t> gmail.com>
   Subject: [Histonet] Nova Red/VIP substrate
   To: Histonet <@t> lists.utsouthwestern.edu
   Message-ID: <e483362e0507070645359d09c3 <@t> mail.gmail.com>
   Content-Type: text/plain; charset=ISO-8859-1
   Dear All
   Could those of you in the community who have used these reagents from
   Vector Labs for double labeled IHC reactions please comment on their
   ease of use & contrast when used in conjuction with DAB substrate.
   Louise Renton
   Bone Research Unit
   University of the Witwatersrand
   South Africa

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