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> |
Reply-To: | |
Content-Type: | text/plain; charset="us-ascii" |
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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