Dear John,

Please could you tell me what the specimen & chamber temperature are? Are
you allowing enough time for the the specimen to reach cutting temperature
after being frozen in liquid
nitrogen-cooled isopentane, as from the information you reported the
embryos show a classic case of being to cold.

Best Regards

Alan Bright

Bright Instrument Co.Ltd.
St Margarets Way
Huntingdon
PE18 6EB
England

Tel No:+44 (0)1480 454528
Fax No:+44 (0)1480 456031
Email: AlanBright@brightinstruments.com
Web Site: www.brightinstruments.com

 +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

-----Original Message-----
From: John C. Dennis [mailto:dennijc@vetmed.auburn.edu]
Sent: 30 November 1999 21:38
To: HistoNet Server
Subject: Cryosectioning and Temp.



Dear Folk

We've been sectioning rat brain (small pieces of rat brain) pretty
well.  But, when we've tried to section embryos (E14-E16) the tissue
crumbles.  These were immersion fixed in 4% paraform. and 0.1%
glutaraldehyde (pH 7.4), cryoprotected in a series of sucrose solutions
(10-30% in PBS), placed in OCT (Sakura) and frozen in liquid
nitrogen-cooled isopentane, and sectioned at the same temperature
(specimen and chamber) as were the brain tissues.

In my limited hands-on experience, I seem to remember that by playing with
the temperatures I could vary the quality of sections.  Or, different
tissues require different temperatures while sectioning.

Is this, or something like this, other people's experience as well?

Or, are there any other helpful hints?

Yours in fragments,

John Carroll Dennis
Anatomy, Physiology, and Pharmacology
109 Greene Hall
Auburn University, AL  36849