[Histonet] Causes of antigen masking (IF on sections vs. flow cytometry)

[ahaines <@t> vims.edu]

This may be in part a philosophical debate, but...

There are some mixed messages in the literature (both scientific and protocol manuals) about the cause of antigen masking.  Most state that it is caused by fixation (for example see below), but others include paraffin embedding as a cause as well.  I rather thought that the problem with embedding might be antigen loss due to heat rather than masking, although with low melting point paraffin it shouldn't be a big contibutor...

Anyway, here's the question:  We are sucessfully doing immunonostaining for an intrancellular antigen on cell suspensions for flow cytometry.  Preparation steps include ~2% paraformaldehyde fixation and permeabilization by methanol.  Next we hope to localize our positive cells within the organ (kidney) by doing immunofluorescence on tissue sections.  Would you agree that, based on our success with staining cell suspensions, we should not expect to encounter problems with antigen masking in the tissue sections?  Is paraffin embedding likely to contribute to antigen loss/masking?  Alternatively, does the thickness of a paraffin section compared to a cell suspension greatly influence the ability to permeabilize?  In theory the ethanol steps in embedding and deparaffinizing should more than adequately permeabilize the cells...

Your thoughts would be appreciated.

Ashley



From: Phyllis Davie <pdavie <@t> phenopath.com>
Date: Fri, 02 Mar 2001 11:54:54 -0800
To: Gerard Spoelstra <spoelstr <@t> numbat.murdoch.edu.au>,
<histonet <@t> pathology.swmed.edu>
Subject: Re: Antigen Retreival Solution

Enclosed please find our methods for antigen retrieval.   We use several different methods.  Please feel free to contact me if you have any questions.
Best of luck,

Phyllis Davie
PhenoPath Laboratories--Seattle, WA
pdavie <@t> phenopath.com

PRETREATMENT, ANTIGEN RETRIEVAL


HEAT INDUCED EPITOPE RETRIEVAL (HIER)

Purpose:  The Heat Induced Epitope Retrieval (HIER) procedure is used to unmask or retrieve epitopes of poorly fixed or over-fixed tissues, as well as unmask epitopes in previously non-reactive tissue.  It is especially useful in formalin fixed tissues.  Formalin fixes tissues by blocking amido groups and by forming methylene bridges between several amino acids in polypeptides.  By this "cross-linking" mechanism, formalin effects a variable number of chemical links not only within a protein molecule but also with adjacent proteins.  These cross-linked proteins are believed to block access of antibodies to their target epitopes, a process often called "masking" of antigens.  Although the exact mechanism by which HIER unmasks epitopes is unknown, many studies have demonstrated improved immunoreactivity of tissue sections following HIER.