Antigen Retrieval Methods

From:Donna Harclerode <>

Good morning Dan,

If there is any tissue available in a paraffin block from 13 years ago?
I would expect better results from a block rather than tissue stored in
formalin for that length of time.  I have successfully stained many
antibodies on very old (30 years and more) paraffin blocks of human and
research animal tissues. I always ran a vimentin (clone V9) control to
be sure the original processing was adequate and not too long in
fixative for IHC. 
Citrate buffer in the microwave was the best the vast majority of my
antigens, but a small number required pH 10 buffer microwave. A few
worked best with no retrieval and the PhraMingen Mouse CD31 clone
MEC13.3 works only with trypsin. 
The vimentin control is an indicator for fixation and processing of
tissues. Vimentin intensity weakens with time in formalin and will
completely disappear eventually. If I can not get staining with vimentin
(using citrate buffer pretreatment), I am skeptical about any staining
with any antibody. 

Good luck

Donna Harclerode HT, HTL (ASCP), QIHC
Lead Scientist
Genset Corp.
La Jolla, CA

 -----Original Message-----
From:   Daniel Martinez []
Sent:   Wednesday, 8 November 2000 12:15 am
Subject:        Antigen Retrieval Methods

     I currently have a rare Parkinson's case that I
am having trouble staining.  This tissue has been
fixing in formalin for 13yrs.  I am attempting to
stain this tissue with several alpha-synuclein
antibodies that we produce.  To date, I have tried
formic acid, proteinase-K, microwave, and boiling
treatments.  Any advice on other methods that might be
worth trying would be appreciated.  Thanks for your

Dan Martinez
CNDR/University of Pennsylvania

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