[Histonet] Her2 Dual ISH and breast processing

[Rene J Buesa]

Val:
Acknowledging that you have a fixation problem is the fundamental step to solving your "staining" problems.
You state that the slices have now a consistent thickness of between 2-3 mm and that is great BUT what about the fixation time?
Thin slices is a first step but until you have the slices properly fixed, you will keep having some problems.
Even when one should never assume, I assume that you are using NBF at room temperature. With that fixative and under those temperature conditions, your 2-3 mm thick breast slices will require 4 hours to be fully penetrated; will require 24 hours to be 100% covalent bound and will require 96 hours to be completely cross-linked.
The 48 hours of maximum exposition to NBF recommended by ASCO-CAP will guarantee a 100% cross-linking of 1 mm thick slices, but your 2-3 mm slices will require 96 hours to be completely cross-linked.
Until you reach an optimum fixation you will keep having sporadic problems of your protocols (depending on greater or lower fat contents of the samples).
René J.

From: "vtolley <@t> cox.net" <vtolley <@t> cox.net>
To: histonet <@t> lists.utsouthwestern.edu 
Sent: Monday, December 10, 2012 11:48 AM
Subject: [Histonet] Her2 Dual ISH and breast processing

Hello all-- 

My lab is having some problems with inconstant results with our Her2 Dual ISH on the Ventana Ultra.  Right now, we know we have a problem with our large breast tissue being under-fixed.  There have been many gripes to the PAs and residents about the thickness of the tissue sections and they have listened.  We are now getting breast sections that are consistently cut between 2-3mm in thickness.  However, we are still having issues with inconsistency in our dual ISH staining.  Many times, the staining is absent.

We are currently processing our breast tissue on a Tissue-Tek VIP6 processor.  Are there any labs out there that are using this processor and also running Her2 Dual ISH on the Ventana with nice, consistent results?  If so, would any of you be willing to share your processing protocol?

Thanks in advance for your help!  



Val



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