Re: Ways to recover ntigens from fixed tissue (paraffin)

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There are various means by which to unmask or retrieve antigens from
fixed tissue.  However, the fixation process denatures proteins and
changes their shape and thus not all antigens are retrievable.  For
those that are, either enzyme digestions or high heat in citrate or EDTA

buffers are commonly used.  High heat may be supplied by means of a
microwave oven, vegetable steamer, or pressure cooker.  If an antibody
requires either enzyme digestion or HIER (heat induced antigen
retrieval), the manufacturer will generally make a note to that regard
on the data sheet.  If not, you can always call the supplier (most have
800 numbers).  For custom made antibodies (not purchased commercially),
you may have to try several methods to see if it is going to work for
you.  The most common buffer is 0.1M citrate buffer at pH 6.0, although
a pH as low as 2.0 and as high as 10.0 have also been used.  Personally
I have never used EDTA for antigen retrieval, so I cannot advise you on
that. The most common enzymes for soft tissues are trypsin, pepsin, and
proteinase K.  For bone matrix proteins in paraffin embedded
(demineralized) bone, the following may be used depending on the
particular protein you are looking for: chondroitinase ABC,
huyaluronidase, keratinase II.  If you have any additional questions in
regard to any of these  procedures, my e-mail address is

Hermina Borgerink, BA, HT(ASCP)IHQ
Wake Forest University Baptist Medical Center
Department of Pathology, Comparative Medicine
Medical Center Blvd
Winston-Salem, NC 27157
ph. 336-716-1538

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