[Histonet] RE: FFPE for Immunohistochemistry.

[Morken, Timothy]

Generally under- fixation in formalin is more of a problem than longer fixation. The reason is first that less fixation does not preserve the tissue and antigens as well (more extraction of proteins in the processing steps), and second, antigen retrieval of any type is more damaging to under-fixed tissue.

Studies have shown that 6 hours is a minimum to achieve formalin cross-linking of peptides and inactivate enzymes. After that time period antigen retrieval will be reproducible. 

Longer fixation is ok. Many studies have shown good antigen retrieval for the vast majority of antigens even after months of fixation when buffer-based heat antigen retrieval is used (as opposed to enzyme digestion). 

An optimum time is probably in the neighborhood of 16 to 24 hours. But  most labs don't do that (clinical labs) and usually 6-12 hours fixation is used.

Her2 and ER/PR guidelines specify minimum 6 hours fixation for bx cores and trimmed tissue.


Tim Morken
Supervisor, Electron Microscopy/Neuromuscular Special Studies
Department of Pathology
UC San Francisco Medical Center




-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Ian R Bernard
Sent: Thursday, April 11, 2013 3:19 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] FFPE for Immunohistochemistry.

Pretreatments are used to recover bonded antigen sites owing to formalin linkage.  What is the optimum or maximum fixation time for tissues that may require Immunohistochemistry staining?

IB
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