RE: Need Antigen Retrieval Help

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From:"Shotsberger-Gray, Wanda" <> (by way of histonet)
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When I HIER, I put all the slides in a 20 slide rack (intended for use on
the Sakura stainer).  the slides with tissue go in the middle of the rack,
and empty slides fill the rest of the slots.  I leave 2 slots in the middle
for the microwave temperature probe.  Then the whole works goes into a
ziplock bag (intended for tissue transport).  I leave a little hole for the
probe to go into.
I "nuke" for 20 minutes at 95 degrees C.  If the stuff boils over, its
caught in the bag, and the bag makes a little humidity chamber that keeps
that from happening, somehow.  I used to have trouble with boil over, but
this cured it.  I have to admit I saw this tip on the histonet a while back.
Barry Ritman may hve posted it originaly...
Wanda Shotsberger
Harris Methodist
Fort Worth TX
From: Bennett, Catherine (Katie)
To: ''
Subject: Need Antigen Retrieval Help
Date: Thursday, November 11, 1999 10:20AM

Hi all!

I'm starting a double staining IHC run this morning that is intending to
take two days, and I may have already committed a fatal error on step one,
so I'm wondering if I should continue or start over.

I am doing microwave antigen retrieval and had a problem with the solution
boiling over and completely out of the coplan jar.  My protocol calls for
microvaving slides in a coplan jar of antigen retrieval for 2x 5 min, but
after just 1.5 min all the solution is boiled over and gone.  Needless to
say, I didn't catch it in time and the slides may have gone through some
microwaving without any solution in the jar.  I had placed the coplan jar in
a small dish of shallow water, so the microwave was very humid at least.

I'm guessing the microwave is much more powerful than the one used in the
protocol I am using and I should experiment with which power setting I
should use.  However, in the meantime, are the slides from today toast
because they have been "cooked" without being in the antigen retrieval
solution, or can I continue with them?

Any suggestions from others on preventing boil-over would also be much

Catherine "Katie" Bresee Bennett
Sr. Technical Associate
Lovelace Respiratory Research Institute
Albuquerque, New Mexico

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