Re: free floating sections

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From:Donna Simmons <> (by way of Marvin Hanna)
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Hi Cynthia - in 'polly-freezing' Montana!
	We routinely stain free-floating frozen sections of perfusion-fixed
rat brain and nerve from 15 to 40+ microns thick -- mostly immuno, but have
done silver techniques, etc. on frozen and vibratome sections.
"Free-floating sections" is clearly the best way to go for optimal
penetration and even staining of thick sections.  We use very dilute
solutions of rare antibodies and stain over the weekend in the cold for
good labelling with very low background.  After staining our sections are
mounted from buffer onto slides using a fine artists' brush, then air dried
before coverslipping with aqueous or xylene based mountant, depending on
the stain/label.
	For IHC on small sets of sections, lidded, stackable styrene tissue
culture wells are very handy for storage after cutting and for the entire
staining procedure.  They come sterile, individually wrapped, in various
configurations of different-sized wells per tray at about $1+ each.  We use
24 well plates for rat brain [4-5 sections/well], and have used the larger
sized wells of 6 well plates for rinsing or for large sections [e.g.,
rabbit or monkey brainstem/cerebellum].   TC plate reusability is a nice
feature for section storage and processing through buffers, etc.  They're
cheap enough to discard if used for antibodies or toxic materials.  Note:
NOT to be used for high % alcohols or other solvent solutions!
Sections can be moved well-to-well with a camel hair brush, wood stick,
fire-polished pasteur pipet made into a hook, etc.-- take your pick
depending on worry about section damage and possible contamination by
carryover of solution from one well to the next.
	We process large numbers of sections in order, and have custom made
plexiglass trays containing drilled wells of desired size with nylon net
bottoms and fitted rinse trays, so that we can use minimum amounts of
precious antibodies.  We put sections in these, and move them all at the
same time from one solution to the next -- wonderfully convenient and best
for staining consistency.
	For net bottom trays in the same size and configuration as tissue
culture wells: contact Lonnie Nason at Nason Machine in Fort Bragg CA
(707/961-0646).  I can fax you a flyer with picture and description, if you
like.  Perhaps you have a local machine shop that can fabricate 'staining
nets' to your own specifications?  For very large sections [whole monkey
brain], Brain Research Labs has nice circular nets of various sizes, that
use crystalizing dishes for solution trays (617/965-5544;  They also sell replacement netting and the glue to
attach it.
	If you're doing stains involving many solutions or solvents that
must be in glass, then small petrie or crystallizing dishes and individual
sections moved by glass hook will do.

Warm thoughts from Sunny California!  -Donna

PS: Advisable to use a rotator tray for long incubations, or at least to
gently hand agitate [swirl] for a minute to assure sections are free
floating and evenly exposed to each solution.

>Date: 25 Jan 2000 17:53:12 -0600
>From: Cynthia Favara <>
>Subject: free floating sections
>	I have been asked to stain free floating sections and am enlisting
>my greatest resource. If possible we would like to use frozen tissue and
>thick sections. The premise is that we can stain without the tissue ever
>drying. Advice from anyone with experience would be greatly appreciated.
>I'll take any ideas and promise not to be offended by sarcasm etc [polly
>would definitely freeze here today]
>Cynthia Favara
>Rocky Mountain Laboratories
>903 S 4th Street
>Hamilton, MT 59840
>ph: 406-363-9317
>FAX: 406-363-9286

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