Hi Marg

Welcome to the club! When we gave the manufacturer of the Herceptest a bad
time for their protocol (because it has been developed at sea level where
water boils as close to 100 C as possible) they were not very enthusiastic
about our comments (I'm stupid enough to live at a higher altitude). We've
tried almost everything under the sun using waterbaths, but were unable to
maintain 95 C for the buffer INSIDE the coplin jar, although we managed to
have this temperature for the waterbath, but that's not the point. Now, as
we do not live in FDA-country (even though I'm sure that the new president
of the USA - if there ever will be one -  will within short declare FDA
rules to be the standard for the whole known world, i.e. the USA), we
modified the manufacturer's protocol and on an on-site inspection, the Dako
rep approved of our method - and a couple of months later asked for our
protocol ... Basically we are boiling the slides directly in the buffer,
using a Bunsen burner (which already gave me a couple of nasty comments on
the histonet on an earlier occasion). However, if you're living at too high
an altitude, where water boils at less than 95 C, our method will not solve
your problem. Insist with the manufacturer that they solve your problem (and
don't accept sending out the slides to a lab at sea level as a solution).
I'm including the protocol for you (but not the histonet) as a PDF file.
Good luck.

Andi Kappeler
Institute of Pathology, University of Bern, Switzerland