RE: FW: Slide storage; Architecture collapse!

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From:"Patterson, Noelle" <PattersonN@NMRIPO.NMRI.NNMC.NAVY.MIL> (by way of histonet)
To:histonet <histonet@magicnet.net>
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See below.

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 2:10 PM
	To:  Patterson, Noelle
	Cc:  rkline@emindustries.com; Histonet
	Subject:  RE: FW: Slide storage; Architecture collapse!

	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.
Wedges are not the problem.  It is the needle core biopsy, and they are
certainly NOT too thick (about 5mm long and the width of the inside of a 20
gauge needle).

	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.
I would need to store these for maybe even months.  I am still developing
procedures for additional surface markers, apoptosis, and cytokine studies.
I will need to go back to any and all samples they give me from surgery and
post-operative tracking/surviving animals.


	Maybe another histonetter can put more input into the immuno side.
I could use this.

	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>>







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