RE: 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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Cynthia,

I am definitely leaning towards cutting all the sections at once, but have
never stored frozen section slides any way besides air dry at room
temperature, protected from dust, after full course acetone fixation.  This,
I know, doesn't work well for the monkey tissue at all.

I think the OCT starts to infiltrate with the water in the kidney while it
is in the cryostat and causes structural distortions.  This is just a guess.
I think the kidney is more susecptible to compression by the temperature
changes, and more apt to have diffusion/exchange of ions and experience
strange gradients that affect the architecture, purely because of its design
for its function.  Again, a guess.

I worry that sucrose cryoprotection will take too long for some of our
needs.  I also worry that it is not a good idea for kidney biopsy (~20 gauge
core needle biopsy usually no longer than 5 mm).  I would hate to lose
sample, or increase handling by adding this step unless it was really worth
it.  But, I would like to give it a try on a nave kidney where I have
several biopsies (limited only by my patience) to see how it goes.  What is
the procedure?  Remember, I have never done anything like this; just a
researcher thrown into histology.  Luckily, I rather enjoy it.

I look forward to "hearing" from you.  Thanks for your help,

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


	----------
	From:  Cynthia Favara [SMTP:cfavara@atlas.niaid.nih.gov]
	Sent:  Thursday, January 28, 1999 5:18 PM
	To:  'Patterson, Noelle'; Histonet
	Subject:  RE: Slide storage; Architecture collapse!

	Noelle,
		I would be inclined to cutt all sections at once and store
for later
	staining. Just my personal preference. I am curious what thoughts
are as to
	why the deterioration is only in kidneys. Have you ever had any
problems
	with brain???
		I am doing more and more sucrose cryoprotection of fixed
specimens
	nad would be happy to share my protocol if you wish. Good luck.
	Cynthia Favara
	NIAID/RML/LPVD
	903 South 4th Street
	Hamilton, MT 59840
	PH: 406-363-9317
	FAX: 406-363-9286


	-----Original Message-----
	From: Patterson, Noelle
[mailto:PattersonN@NMRIPO.NMRI.NNMC.NAVY.MIL]
	Sent: Tuesday, January 26, 1999 3:11 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 '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
	pattersonn@NMRIPO.NMRI.NNMC.NAVY.MIL




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