[Histonet] IF Protocol on skin

Gagnon, Eric gagnone <@t> KGH.KARI.NET
Fri Feb 13 09:44:49 CST 2009

Hi Gudrun, 
We've rarely done Ig's on FFPE renal sections on the Ventana -  our renal pathologist prefer these on IF frozen sections only.  We do have the IgG, IgA, IgM, IgE antisera for lymphoma cases.  We use Dako Antibody Diluting Buffer, Ref. S0809 to dilute our FITC antisera.
As I said, we didn't persist with validating these antibodies for use with renal or skin IF's on the XT, mainly due to our pathologist's satisfaction with the manual IF staining method.  This was back in January 2006, with the antibodies incubating for 4-8 minutes.
Hope this helps,


From: Gudrun Lang [mailto:gu.lang <@t> gmx.at]
Sent: Thu 2/12/2009 10:41 AM
To: Gagnon, Eric
Subject: AW: [Histonet] IF Protocol on skin

Hi Eric,
thanks for your response. I have several further questions.
Did you stain frozen renal sections in the benchmark? Or did you also try
NBF-fixed tissue?
What diluent do you use for the fitc-antisera? We have also the
ventana-machine. And I consider to use their antibody-diluent for this.

This week I have stained NBF-fixed skin (Pemphigoid) with IgG, IgM, IgA in
the Benchmark XT. The IF of the frozen part showed IgG and C3C linear
staining. The IHC of the FFPET showed very strong staining with all
Immunoglobulins. Perhaps we can find a diagnostic value.


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Von: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] Im Auftrag von Gagnon,
Gesendet: Mittwoch, 11. Februar 2009 22:03
An: histonet <@t> lists.utsouthwestern.edu
Betreff: [Histonet] IF Protocol on skin

Hi Gudrun,

Our IF protocol is very similar to yours...we use 1/30 dilutions as well,
although we rinse before the FITC-antisera (Dako) are applied, with frozen
sections cut at 5 microns. 

We did experiment with this procedure on our BenchMark XT's, with some
success.  The pathologist who reads most of renal biopsies IF's decided that
our manual method was "robust" enough (i.e. foolproof enough) that we could
continue using it.  Manual staining saves us trying to fit in a new or
current run when the XT is already running.

Regarding our dilutions, we make 3 ml at a time, and have not had any
problems with the staining diminishing before the antisera are used up.

Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada

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