Noelle,

By sections,  I meant tissue samples.  If the wedge is too thick it will
not freeze uniformly which would cause freezing artifact.  You may want to
bi-sect (did I spell that right) the wedge, if you can.

Whirlpak bags can probably be purchased from many distributors.  Two, I
know for sure are VWR or Fisher.  Slide boxes are plastic and can be 25 or
50 capacity.   Also, very easily available from many distributors.

The freezer can be a -70.  You can cut sections on glass slides and put
them in a -70 freezer.  Some people use a desicant or a silica gel to
prevent moisture.  If I remember correctly, the immuno's where cut the day
before they where stained.  Extra slides for muscle biopsies were always
taken and stored in case additional stains were required without losing
enzyme activity.

Maybe another histonetter can put more input into the immuno side.

Hope this gives you some ideas.

Rande Kline HT (ASCP)
Technical Services
EM Science




"Patterson, Noelle" <PattersonN@NMRIPO.NMRI.NNMC.NAVY.MIL> on 01/29/99
09:56:26 AM

To:   rkline
cc:   Histonet <HistoNet@Pathology.swmed.edu>
Subject:  RE: FW: Slide storage; Architecture collapse!




Do you mean that you cut sections, put on glass slides, and just return
them
to the freezer?  What freezer do you store them in?  What kind of slide
boxes (plastic, wood, slide mailer, does it matter)?  I do the same for
preparation and storage (well, instead of whirl bags I use saran wrap).  By
whirl bags, do you mean the biohazard transport bags?  Where can they be
ordered?  I am getting tired of saran wrap, though it is cheaper than whirl
bags (I suspect).
Noelle Patterson
Naval Medical Research Center
Bethesda, Md
pattersonn@NMRIPO.NMRI.NNMC.NAVY.MIL


     ----------
     From:  rkline@emindustries.com [SMTP:rkline@emindustries.com]
     Sent:  Friday, January 29, 1999 8:56 AM
     To:  Patterson, Noelle
     Cc:  Histonet
     Subject:  Re: FW: Slide storage; Architecture collapse!

     Noelle,
      Could it be freezing artifact your seeing?  Are the sections thin
enough
     to freeze quickly?

     I used to freeze in isopentane with OCT. Kept frozen specimen on
cryostat
     chuck or on  cork platforms sliced from stoppers.  Stored in -70
freezer
     wrapped in aluminum foil in whirl-bags.  Filed whirl-bags in small
boxes
     numerically and by year.  Whirl-bags worked great.

     After cutting returned to bags wrapped in foil.  Never had a
problem.

     Used to store cut slides in slide boxes that hold 50 slides.

     This was done for muscle bx's and immuno's.

     Rande Kline HT (ASCP)
     Technical Services
     EM Science




     "Patterson, Noelle" <PattersonN@NMRIPO.NMRI.NNMC.NAVY.MIL> on
01/28/99
     11:36:53 AM

     To:   Histonet <Histonet@pathology.SWMED.edu>
     cc:
     Subject:  FW: Slide storage; Architecture collapse!




     Maybe it will get through this time.  If this is a repeat, please,
someone
     email me directly.  I am only occasionally getting histonet mail,
and have
     not yet received my post (this is my 3rd time).  I would like to
know who
     to
     contact about this, our server people or histonet....could be either
since
     I
     am getting some outside mail.

     Noelle Patterson
     Naval Medical Research Center
     Bethesda, Md
     pattersonn@NMRIPO.NMRI.NNMC.NAVY.MIL


     ----------
     From:  Patterson, Noelle
     Sent:  Tuesday, January 26, 1999 5:09 PM
     To:  'Histonet'
     Subject:  Slide storage; Architecture collapse!

     I am curious about how people section frozen (fresh tissue snap
frozen in
     OCT in an isopentane/dry ice bath) tissue and then store it for
later
     analysis (H&E, immunohistochemistry, etc.).  I have generally been
able to
     cut the sections I need, stain them and store the block at -70
degrees C
     until I need it for further analysis.  The repeat freeze-thaws that
occur
     (between storage and cutting of the blocks) has never caused any
notable
     architecture demise/artifact, until I began cutting kidneys.  These
     tissues,
     even in the 2 round of cutting, have distended tubules and what look
like
     water-filled nuclei...in general making the architecture look
horrible,
     without harming the immunoreactivity.

     Recent discussions have dealt with 1) cryopreservation in sucrose
prior to
     OCT embedding (which I have considered, but would need to do this
routinely
     with needle core kidney biopsies.  This seems to be impractical, and
I
     worry
     about the immunoreactive epitope stability) and 2) cutting all the
sections
     needed and more, quick fix in acetone, then store in a freezer until
IHC,
     etc.

     This second point sounds interesting to me, but I need to know if it
will
     preserve the original architecture (to that I see when it is first
thawed
     to
     -20, cut, fixed, and stained) while stored in the freezer (or does
storage
     in this manner introduce its own architectural artifacts).  At what
     temperature and how do you store the slides after cutting?  What do
you
     usually "quick fix" in and for how long?  I generally fix for 10 min
in
     -20C
     acetone prior to IHC on freshly cut sections, so should I use
acetone?  For
     H&E's I fix in 10% NBF, should these be quick fixed in formalin.
What are
     the storage conditions? (where to order the dessicant, what kind of
     containers are the slides stored in, how long can these slides be
stored
     before architecture is notable different, how does one treat them to
get
     them ready to stain again).  I know a number of these answers have
been
     discussed, but I haven't saved them.  Unfortunately, it wasn't until
the
     end
     of the discussions that I realized they may apply to what I do and
improve
     the work!  I hope you don't mind repeating yourselves, as I have not
seen a
     summary on these issues posted.

     The basic question:  How can I keep the morphology of my kidney
biopsies
     (and to a lesser extent, kidney wedges) from collapsing during
freeze-thaws
     (-70 - -20C)?  This effect is dramatic on the third thaw of mouse
kidney
     (divided into <<File: ATT32345.txt>>