[Histonet] RE: [IHCRG] HRP-labeled primary antibodies

[Tony Henwood]

Kim,
 
The direct IPX is the easiest method apart from a direct
immunofluorescence. Very few steps:
1.    Block endogenous enzyme
2.    Block non-specific Ab binding
3.    Put the HRP-conjugated Ab on (a an appropriate dilution, usually
more concentrated than if you were using an ABC or Polymer amplification
method)
4.    Incubate in your usual DAB-H2O2 solution
5.    Counterstain and you done.
 
Throw in a few buffer washes in between and maybe some antigen retrieval
if required.
 
We use the direct IPX method for renal biopsies (IgG,M,A etc). See
Birchall, I.W., (1996) "Direct antibody method for formalin fixed,
paraffin embedded renal biopsies: a comparison with the peroxidase
labelled streptavidin/biotin method" J. Histotechnol 19(2): 125-129.

Regards 

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC) 
Laboratory Manager & Senior Scientist 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


	-----Original Message-----
	From: ihcrg <@t> googlegroups.com [mailto:ihcrg <@t> googlegroups.com] On
Behalf Of Kim Merriam
	Sent: Thursday, 21 May 2009 10:42 PM
	To: Histonet; ihcrg <@t> googlegroups.com
	Subject: [IHCRG] HRP-labeled primary antibodies
	
	
	Hi All,
	 
	Has anyone had experience doing IHC on FFPE tissues with
HRP-labeled primary antibodies?  I was wondering what the best way to
detect them would be.  I assume that going strait to DAB would not work,
since no amplification is there.  I was thinking of using a biotinyl
tyramide step to amplify the signal.
	 
	Also, do you think the final antibody concentration would need
to be higher than with traditional, unlabeled primaries?
	 
	Thanks,
	Kim
	 
	Kim Merriam, MA, HT(ASCP)QIHC
	Cambridge, MA 



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