RE: sectioning of frozen tissues

From:Tony Henwood

RE: sectioning of frozen tissues


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

-----Original Message-----
From: Dale Osborne []
Sent: Thursday, 19 December 2002 7:34
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
          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

    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

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