Forwarded: RE: FW: Slide storage; Architecture

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From:"Karen D. Larison" <LARISONK@UONEURO.uoregon.edu> (by way of histonet)
To:histonet <histonet@magicnet.net>
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Histonetters,

I have a question related to Noelle's.  I recently found a paper (Peptides,
7,
155-159, 1986) describing a method for long-term storage of fixed blocks or
free-floating sections in an ethylene glycol-based cryoprotectant at -20C.
The
method preserves the brain tissue well enough that the authors were able to do
obtain good cellular morphology on the EM level.  Does anyone routinely use
this method?  I have one researcher here who is trying the method as it would
make his life so much easier.  But he is interested to know if others are
routinely using this method.

Thanks.

Karen Larison - University of Oregon




Date:          Fri, 29 Jan 1999 09:56:26 -0500
From:          "Patterson, Noelle" <PattersonN@NMRIPO.NMRI.NNMC.NAVY.MIL>
Subject:       RE: FW: Slide storage; Architecture collapse!
To:            rkline@emindustries.com
Cc:            Histonet <HistoNet@Pathology.swmed.edu>

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>>




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