Re: sectioning of frozen tissues

From:Patsy Ruegg

I can only reintegrate Chris V.  In my own experience I have never seen lost
immunoactivity from air drying FS overnight, but as Todd mentions, maybe I
have not yet tested for a protein expression which may be adversely impacted
by this drying, or maybe soaking in buffer as I do after fixation is enough
to rehydrate and reverse the drying effects.  And so goes the magic of IHC
for me.
Patsy Ruegg

----- Original Message -----
From: "Todd Sherman" 
To: "HistoNet Server" 
Sent: Thursday, December 19, 2002 3:42 AM
Subject: Re: sectioning of frozen tissues

> 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
> Todd Sherman
> President
> HistoSoft Corporation
> -----------------snipped from digest-----------------------
> Date: 18 Dec 2002 14:45:44 -0600
> From:
> 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
> ----------------------------------------------------------------------

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