Just off the top of my head, Vanessa, I would look at the temperature of
your chamber and/or object holder.  Different tissues have different optimum
cutting temperatures.  As to your freezing method, I would suggest either a
commercial freezing bath used with isopentane (2 methyl butane) or making
your own freezing set up by using a stainless steel beaker of isopentane
cooled by liquid nitrogen.  Either of these methods will result in less ice
crystal formation than freezing in the cryostat.  Instrumedics also makes
the "Gentle Jane" freezing apparatus that does a good job as well.

As to sectioning, I would steer clear of freezing sprays especially if
you're subsequently doing immunohistochemistry on the sections.  I don't
quite understand your comment about freezing the tissue before sectioning;
we are talking about fresh frozen tissue as opposed to fixed-frozen or
paraffin-embedded tissue are we not?  Therefore your tissue is already
frozen prior to sectioning. Otherwise, all the things you're trying seem
logical; I would just make sure everything that you can think of is tight.
One little thing being even slightly loose can wreak havoc.

Hope some of this is of some help; hopefully other histonetters will have
more suggestions.

Linda A. Sebree, HT
University of Wisconsin Hospital & Clinics
IHC/ISH Laboratory, D4/218
600 Highland Ave.
Madison, WI 53792-2472
(608)265-6596
FAX: (608)263-1568
<la.sebree@hosp.wisc.edu>


-----Original Message-----
From: Vanessa Heim [mailto:heimvk@bu.edu]
Sent: Thursday, December 07, 2000 6:20 AM
To: histonet@pathology.swmed.edu
Subject: Cryostat sectioning


Thank you to everyone that has repsonded to my problem of sections
falling off the slides during staining.  I actually have another problem
that I have been encountering quite frequently...I seem to have trouble
consistently getting great sections.  Some days the sections are
beautiful and otherdays I destroy the tissue block just trying to get a
few good sections.  We are using a cryostat machine now and was
wondering if anyone had some advice for me on this issue.  I have tried
changing the angle of the knife, I have changed the blade on several
occasions..I even freeze the tissue before sectioning and use a freeze
spray during the procedure to keep the sample cool.  Is there a special
way to freeze the samples before cutting or protocols available
specifically for processing/freezing tissues specifically for the
cryostat???  I have looked online and haven't come up with anything too
substaintial.  Like I said earlier this is all  new to me so any help
would be greatly appreciated.


Vanessa Heim
Boston University Medical Center
Research-Dept Urology/Surgery
Boston VA Hospital
Boston, MA  02130