"Dr. Ian Montgomery." <ian.montgomery@bio.gla.ac.uk>
<html>
Su,<br>
<x-tab> </x-tab>Let me
offer some advice before bowing to John's greater knowledge. <br>
<x-tab> </x-tab>Using
acetone at -80C you are freeze substituting the tissue. Depending on size
of tissue sample this can take many days - remember, small is beautiful.
If possible use a rotator in the freezer or frequently agitate the
tissue. Warm up slowly and evenly, probably over a day. <b>Do not use any
solvents with water, your tissue is dehydrated so why hydrate again.</b>
Methyl benzoate, mmmmm, wonder why, it is a clearing agent, not my first
choice, but can be used to make tissue less brittle. Xylene is used here
as a linking agent between methyl benzoate and wax. <br>
<x-tab> </x-tab>Depending
on the tissue and size I'd be tempted, in the first instance, to have a
trial. Wring the changes and see what happens, but definitely, <b>no
water.<br>
</b>Ian. <br>
<br>
<br>
<blockquote type=cite class=cite cite>Date: Fri, 30 Mar 2001 07:51:22
-0500<br>
From: "Su, Phy-Huynh" <psu@shctampa.usf.edu><br>
To: "'J. A. Kiernan'" <jkiernan@uwo.ca>,<br>
"'Histonet'"
<Histonet@pathology.swmed.edu><br>
<br>
Dear Histonetters:<br>
<br>
May I ask a question about methyl benzoate, please.<br>
<br>
I have received a protocol to fix tissues in acetone, at -80 degree C
with<br>
few changes, then bring the tissues up to RT in a fresh one, then going
into<br>
methyl benzoate before going into xylene, then paraffin. I
understand that<br>
methyl benzoate is a clearing agent, as xylene, but is it really
necessary?<br>
Does it act as another clearing agent here or does it have any
other<br>
affects? Can I just go directly to Xylene as routine
processing? How about<br>
putting the tissues into 50%, 70%, 100% alcohol? Will this
deterioate the<br>
tissues?<br>
<br>
Thank you.<br>
Su</blockquote>
<x-sigsep><p></x-sigsep>
<font color="#0000FF">Dr. Ian Montgomery,<br>
West Medical Building,<br>
University of Glasgow,<br>
Glasgow,<br>
G12 8QQ.<br>
Tel: 0141 339 8855. Extn:6602.<br>
Fax: 0141 330 2923<br>
e-mail: ian.montgomery@bio.gla.ac.uk</font></html>