Re: sectioning of frozen tissues

From:Todd Sherman

Hello Dale,

The drying of sections overnight can impact the immunoreactivity of some proteins and only by empirical data can you determine the 
degree.  The tertiary and quaternary structure of some proteins will be altered should their hydrophilic regions be devoid of an 
aquatic environment.  If an antibody is directed at a particularly hydrophilic region, it may not bind if the protein's structure becomes 
too non-native, ie. denatured.  It is the fixation that helps (among other things) maintain tertiary and quaternary structure in the 
absence of a saturated, aqueous environment.

If the tissue can be rehydrated at some point prior to exposing the antigenic sites to a site-directed antibody, the effect of the 
dehydration process would likely be minimal.  That is because the primary and secondary protein structure is retained despite the 
dehydration process.  Re-introduction of water will cause normal thermodynamics to return and the protein will renature into its most 
energetically favorable position assuming other chemical fixatives have not changed the original structure excessively.

Tissue type (which, in this context, equates to concentration of antigen)  and antibody site-specificty are all major factors that come 
into play when determing the effect of dehydration on IHC.  Further, the antibody is paramount.  As long as it can contact the original 
amino acid sequences for which it was designed, detection should be acceptable.  Again, only through repeated experimentation or 
complimentary observations by others can you know if the particular antigen that you are trying to visualize be detrimentally impacted 
by dehydration.

Hope this helps,

Todd Sherman
HistoSoft Corporation

-----------------snipped from digest-----------------------
Date: 18 Dec 2002 14:45:44 -0600
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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