| From: | Tony Henwood |
Dale,
Several pointers I've found:
1. Freeze the tissue as fast as possible (eg liquid nitrogen)- more morphology is usually due to this.
2. Try ethanol fiaxation (30min) - take via graded alcohols to water for IPX - do not let dry.
3. Air drying 20min, fix in cold acetone (from -20oC), allow acetone to reach room temp before removing slides to air dry. Then into buffer and IPX.
This works with many routine antibodies, some (eg neuroblastoma) work on air dried, others only with ethanol fixation, others it doesn't seem to matter.
Hope this is helpfull
Tony Henwood JP, BappSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager
The Children's Hospital at Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: (02) 9845 3306
Fax: (02) 9845 3318
http://www.histosearch.com/homepages/TonyHenwood/default.html
http://us.geocities.com/tonyhenwoodau/index.html
-----Original Message-----
From: Dale Osborne [mailto:oskaki@morpheus.wustl.edu]
Sent: Thursday, 19 December 2002 7:34
To: histonet@pathology.swmed.edu
Subject: sectioning of frozen tissues
Greetings histology experts,
We are doing immunohistochemistry on frozen sections but so many
times the morphology of the sections (especially small tissues such as
mouse thymus) are terrible. Consequently we've been looking for
alternative protocols. We are considering the protocol listed on the
Pharmigen web site. Part of the protocol is as follows:
II. Sectioning of Frozen Tissues
1.Before cutting sections, allow the temperature of the block
to equilibrate to the temperature of the cryostat (-20#176#C).
2.Place the tissue block on the cryostat specimen disk. Adjust
the positioning of the block to align the block with the knife blade.
Cut tissue block until the desired tissue is exposed.
3.Cut sections of the desired thickness (usually 5 um), place
the sections on a Fisher Superfrost slide and dry overnight at room
tem-perature.
4.Fix slides by immersion in cold acetone (-20#176#C) for 2
minutes or other suitable fixative (e.g. alcohol, formal alcohol,
formalin, etc.), air dry at room temperature and proceed to staining
(Section III).
5.Alternatively, the frozen section slides can be stored for a
short period of time at -70#176#C in a sealed slide box. When ready to
stain, remove
slides from freezer and warm to -20#176#C in the cryostat or
-20#176# freezer, fix for 2 minutes in cold fixative (acetone or other
suitable fixative)
and allow to come to room temperature to continue with the
staining.
Now, my question is wouldn't allowing the section to dry overnight
at room temperature (as described in step 3) result in destruction of
certain proteins (maybe ones we want to stain for)?
Thank you in advance,
Dale Osborne
Washington University School of Medicine
Anesthesiology Dept.
St. Louis, MO
**********************************************************************
This email and any files transmitted with it are confidential and
intended solely for the use of the individual or entity to whom they
are addressed. If you are not the intended recipient, please
delete it and notify the sender.
Views expressed in this message and any attachments are those
of the individual sender, and are not necessarily the views of the
Childrens Hospital at Westmead
This footnote also confirms that this email message has been
virus scanned and although no computer viruses were detected,
the Childrens Hospital at Westmead accepts no liability for any
consequential damage resulting from email containing computer
viruses.
**********************************************************************