Nicola,

Try bringing your slides in the buffer just up to a bare simmer in the
MW and then transfer to a waterbath at your desired temp. (we used 92
-94 degrees C) for 20 - 40 minutes.  Follow with your cool down and
proceed with your staining.  Before we had decloaking chambers (pressure
cookers) we found this method to be the gentlest way to retrieve.

Linda A. Sebree
University of Wisconsin Hospital & Clinics
IHC/ISH Clinical & Research Laboratory
DM223-VA
600 Highland Ave.
Madison, WI 53792
(608)265-6596
FAX:  (608)262-7174



-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Nicola
Cragg
Sent: Friday, July 08, 2005 4:41 AM
To: Histonet@lists.utsouthwestern.edu
Subject: [Histonet] HIER - best retrieval with minimum tissue damage?


Hello,

I'm a big fan of microwaving in citrate buffer as a method of heat
inducing epitope / antigen retrieval.  However, I do run into problems
when microwaving skin.  I've done a lot of IHC on skin and have found
the microwaving is great when staining antigens in the epidermis, but I
do have problems if the antigens I want to look at are in the dermis and
require microwaving / heat for retrieval.  The tissue is often damaged
and the morphology of the dermis is disrupted, so when I do see evidence
of specific staining it's difficult to be confident in it.  

Obviously, when optimising antibodies, I simultaneously attempt it
without antigen retrieval or via other methods such as proteolytic
digestion, but some antigens in the dermis appear to require heat.  I
have tried steaming, as I've read this is a gentler method, but I found
it to be more damaging than microwaving.  I have had some experience
with using a pressure cooker and a waterbath for HIER but have not yet
tried it specifically for skin.  I use APES coated slides.  Does anybody
have any suggestions?

Kind regards,

Nicola Cragg
Histology & IHC
EpiStem Ltd
Manchester, UK
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