Dear Vincenzo,
You didn't describe the fixative used for your frozen sections, but I 
assume that it was acetone. However, as you are trying to stain a 
nuclear marker it is strongly advised to use either Zamboni, 4% 
paraformaldehyde or just buffered formalin for 5 min at room 
temperature. Then wash with PBS (or TBS) and start your IHC procedure. 
Most nuclear markers get diffuse after acetone fixation.
Furthermore, you described the application of different antigen 
retrieval methods. To my opinion, antigen retrieval is not needed for 
frozen sections because antigens/epitopes are not hidden due to 
fixation or something like with FFPE sections. Even if buffered 
formalin is used as fixative for frozen sections there is nothing to 
retrieve, simply because a 5 min fixation time is far too short to 
cause cross-linking of proteins. And indeed as you described, the 
tissue morphology of frozen sections will heavily suffer from antigen 
retrieval procedures. 

Good luck,
Chris van der Loos
Dept. of Cardiovascular Pathology
Academical Medical Center
Amsterdam - The Netherlands

>Original Message ----- 
>From  "Vincenzo La Manna"  
>Date  Tue, 16 Sep 2003 15:48:43 +0100 (BST) 
>To   
>Subject  [Histonet] immunohistochemistry and frozen sections 
>
>Hi Histonetters,
>
>I've been struggling for 4 months on my 5 microns
>frozen sections trying to find estrogen receptors.
>I've tried both immunoperoxidase kit from Vector
>laboratories either indirect immunofluorecsence (FITC
>Goat anti mouse Secondary Ab from Sigma) but I've not
>yet obtained good results.
>My primary Ab is a mouse monoclonal to bovine
>Estrogen receptor from Cymbus biotechnology.
>Sections come from tissue that is supposed to be a
>non-target tissue (mid laminar region from cow's
>hooves)blocking with 2% goat normal serum (sigma)
>whash in tbs buffer
>All different methods for antigen retrieval have been
>tested but sections look poor regarding histologic
>quality and I still have very big problems with non
>specific binding an staining (indirect
>immunofluorescence).
>It looks like frozen sections are too tender to
>efford the whole processing.
>Every suggestion or reference is welcome,
>thank U
>
>La Manna Vincenzo
>Department of Agriculture and Forestry, University of
>Aberdeen, 581 King Street, AB24 5UA, Aberdeen , UK.
>Telephone; 01224 274259
>Fax; 01224 273731
>e-mail v.lamanna@abdn.ac.uk




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