Date sent:      	Thu, 11 May 2000 15:09:18 +0800
From:           	muhaizan <muhaizan@mail.hukm.ukm.my>
Subject:        	Immunohistochemical stain for immunoglobulins and complements
To:             	histonet@pathology.swmed.edu

> Can anybody give his/her opinion on how to optimise immunohistochemical
> staining for immunoglobulins and complements on renal tissue. My effort is
> still doesn't come to success eventhough I've tried various dilution of
> primary antibody and various timing for the antigen retrieval method. Non
> specific staining especially for the endothelial cells is still there.

 If you are getting reactivity in (or on) endothelial cells then it is 
likely that you are simply detecting circulating Ig and /or 
complement.This could be confused with actual basement 
membrane Ig and hence with an immune complex disorder (e.g. 
IgA nephropathy). To the trained/experienced eye this should not 
cause confusion with real disease but the technique must be 
adjusted to eliminate what is a specific but not appropriate reaction.
In my experience renal ICC requires a different approach with 
regards to antigen retrieval in order to eliminate this "artefact". I 
have always used 0.05% trypsin pH7.8 @37oC for 60-65 mins 
which will remove any circulating Ig leaving any Ig complexes on 
the basement membrane available to your antisera. The rest of the 
technique is standard and antibody dilution as recommended by 
the vendor. Try and get hold of a known immune complex disease 
as your control, check with fluorescence if necessary.
It is better to overdigest rather than under as the final result is still 
interpretable despite the fact that you may lose tissue morphology,
a diagnosis is still possible.
As for endogenous biotin, I have never found this a problem 
(negative controls clear) just make sure your bridge and ABC are 
adequately dilute (and the DAB for that matter).
Hope this is of some help,
Terry Hacker,
Medical Research Council,
Harwell,
Didcot,
Oxfordshire, OX11 ORD
01235 834393 x360