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Using 1% or greater H2O2 to block endogenous peroxidase has a detrimental
effect
on the epitope recognised by anti CD4 clone 1F6 (ref. datasheet).
NC recommends blocking with 0.5% H2O2 before unmasking the antigen,
but in my hands, best results with this antibody were obtained when
endogenous
peroxidase was blocked after incubation of primary antibody
(higher concentrations of H2O2 may then be used)
Please take a look at "Blocking Peroxidase", Discussion Board at
http://home.no.net/immuno or go there directly at:
http://home.no.net/immuno2/yabb-bin/yabb/YaBB.cgi?board=news;action=display;
num=1031669926 





Hogne Roed Nilsen
http://home.no.net/immuno
immunohistochemistry@netcom.no




 
Date: 12 Apr 2003 22:10:59 -0600
From: IHC World 
Subject: Re: "Who is the first"


RE: "Who is the first"In theory, blocking of endogenous peroxidase activity
can be applied at any step BEFORE incubation of HRP-conjugated antibody. I
usually do the blocking step after HIER/PIER, but have been testing it
before
HIER/PIER. No difference was observed. So I think the key is to be
consistent
cross all experiments.

Richard
IHC World
Email: info@ihcworld.com
Web: www.ihcworld.com

  ----- Original Message ----- 
  From: Tony Henwood 
  To: 'Fernando Capela e Silva' ; HISTONET@pathology.swmed.edu 
  Sent: Wednesday, April 09, 2003 4:43 PM
  Subject: RE: "Who is the first"


  We block endogenous peroxidase (Methanol-H2O2) before HIER or enzyme
retrieval 

  Tony Henwood JP, BappSc, GradDipSysAnalys, CT(ASC) 
  Laboratory Manager 
  The Children's Hospital at  Westmead, 
  Locked Bag 4001, Westmead, 2145, AUSTRALIA. 
  Tel: (02) 9845 3306 
  Fax: (02) 9845 3318 

  http://www.histosearch.com/homepages/TonyHenwood/default.html 
  http://us.geocities.com/tonyhenwoodau/index.html 




  -----Original Message----- 
  From: Fernando Capela e Silva [mailto:fcs@uevora.pt] 
  Sent: Wednesday, 9 April 2003 18:31 
  To: HISTONET@pathology.swmed.edu 
  Subject: "Who is the first" 



  In immunohistochemistry "who is the first"? 

  Pre-treatment (HIER or enzymatic digestion) of tissues and after hydrogen 
  peroxide to eliminate endogenous activity 

  or 

  Hydrogen peroxide to eliminate endogenous activity and after pretreatment 
  (HIER or enzymatic digestion) of tissues 



  I already see both procedures. Is not important the order? 

  With compliments 

  * * * * * * * * * * * * * * * * * * * * * * * * * 
  Fernando Capela e Silva 
  Laborat#243#rio de Biologia da Conserva#231##227#o 
  Departamento de Biologia 
  Universidade de ...vora 
  Apartado 94 
  7002-554 ...vora 
  PORTUGAL 

  Phone: +351-266 760 800 
  Fax: +351-266 711 231 
  Email: fcs@uevora.pt 

  http://evunix.uevora.pt/~fcs/FernandoCapelaSilva.htm