[Histonet] RE: HRP-conjugated secondary and biotinylated-secondary

From:"C.M. van der Loos"


   The existence of both biotinylated- and HRP-conjuated secondary
   antibodies reflects the development of IHC techniques into
   more staining efficiency/sensitivity. In the old days, a 2-step
   staining method using a HRP-conjugated secondary anti-mouse or
   anti-rabbit was common practice. Mid 80-ies the 3-step staining method
   using a biotinylated 2nd step reagent and streptavidin/HRP conjugate
   or streptavidin-biotinylated HRP complex became very popular because
   of its superior staining efficiency/sensitivity. The disadvantage here
   is the occurrence of endogenous biotin in many tissues. Endogenous
   biotin is not present in untreated formalin-fixed and
   paraffin-embedded tissue sections, but will be retrieved especially
   when using Tris-EDTA pH8-9.

   Only recently we have gone to a 2-step staining procedure again, now
   based on both secondary antibody and HRP attached to a polymer
   backbone. The efficiency/sensitivity of these polymers is comparable
   with the 3-step streptavidin/HRP procedure, but avoids the problem of
   endogenous biotin.

   I hope this small historical overview helps.

   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
   e-mail: [1]c.m.vanderloos@amc.uva.nl

   ----- Original Message -----
   From  Subratab 
   Date  Sun, 30 Jan 2005 0:18:56 +0600
   Subject  [Histonet] HRP-conjugated secondary and

      Is it a common practice to use HRP-conjugated secondary antibody
      instead of biotinylated secondary antibody for immunohistochemistry
      (with DAB developing system)? I am confused about the advantage/
        disadvantage  of  using  HRP-conjugated  secondary  antibody  for
      Subrata Biswas
      UNICAMP, SP, Brazil.


   1. mailto:c.m.vanderloos@amc.uva.nl
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