Slide storage; Architecture collapse!

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From:"Patterson, Noelle" <PattersonN@NMRIPO.NMRI.NNMC.NAVY.MIL> (by way of histonet)
To:histonet <>
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I think histonet address has changed/is case sensitive now.  Is that true?
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 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,

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 's or 1/3's for embedding), and on the 2nd thaw of monkey
kidney biopsies; although I have not noticed any great detriment in monkey
kidney wedges.  I think that cutting through the entire biopsy at one
sitting, putting the tissue on superfrost plus slides, quick fixing and
storing the slides I don't use that day may be the best answer.  I
originally thought that cutting through the biopsy, fixing all slides as
usual, and storing the air- dried slides would work (this always worked for
the mouse tissue).  However, I found that in monkey tissue, the immune cell
markers I am looking at lose staining intensity (i.e. do not remain stable)
even after just one day of air drying.  There must be a better way!

Any and all preferences and ideas are welcome.  Thanks to listening to the
full story.  Sorry to drone on for so long.

Gratefully yours,
Noelle Patterson
Naval Medical Research Center
Bethesda, Md

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