Re: "my" (his) problem ... (long)
|From:||"J. A. Kiernan" <email@example.com>|
On Thu, 10 May 2001, Bonnie Wayne wrote:
> Yes, the gelatin blocks are fixed in formaldehyde ...
> ... hrs to several weeks. The blocks are fixed but not frozen.
That must mean you cut the sections with a vibrating
microtome (Vibratome). They should have excellent
morphology, with little in the way of differential
shrinkage spaces around small blood vessels and large
neurons and, of course, no ice crystal holes.
Such sections are rather thick (whatever the setting on
the machine might say, and the manufacturers admit as
much in their bumph), and it's quite likely that the
gelatin and the brain dry by evaporation at different
rates - hence the curling - the upper surface of the
gelatin shrinking in area and pulling up the edges of
the still-damp under-surface. When lifted off the
slide in this way, the under-surface also dries, and
the edge of the section becomes rigidly set at an angle
to the plane of the slide.
1. Do all the staining etc on free-floating sections
rather than after mounting. For many methods this also
gives better results, because the reagents penetrate the
thick section from both surfaces, not just from the top.
2. I've forgotten what staining methods you said you were
using. If the end-product(s) is/are insoluble in water
(e.g. DAB oxidation product in immunochemistry) let the
sections dry just enough for flattening and then apply
the coverslip using an aqueous mounting medium.
3. If something water-extractable is used (e.g. a basic
dye like neutral red or toluidine blue for Nissl staining),
try to find a water-insoluble substitute. (This might prove
very difficult for a good, permanent Nissl stain.) Making
an aqueous mountant alkaline greatly prolongs the preservation
of stainings by basic dyes.
4. If a permanent mount in a resinous medium cannot be
avoided, dehydrate and clear the unmounted stained sections,
mount them on the slide and then apply the resinous medium
and coverslip. There are various tricks to reduce the
brittleness of the dehydrated sections and prevent (or at
least reduce) cracking when the folds are straightened out.
This is a very labour-intensive procedure; no good for
mass production! If you're really gung-ho about it, ask
for help again. You'll probably get lots of very different
hints and wrinkles (oops! - wrinkle-smoothing hints).
5. While typing Item 4 above, I was recalling the horridness
of processing free floating sections (frozen ones in my case,
but it would be the same with vibratome sections) all the way
into a resinous medium. A light bulb with an exclamation mark
inside suddenly incandesced in my hippocampus, and another
possibility emerged. See Item 6 below. It's a long-winded
paragraph and contains no answer to your problem, but there
are good techniques that allow processing of a dozen or two
slides at a time, with minimal detachment of the tissue.
6. Thick sections are not unlike whole-mounts of dissected
layers of the intestinal wall. These are nice squares that
annoyingly curl up off the slide with drying. A chap called
Richardson published extremely detailed descriptions of how
to handle such specimens, about 35 years ago. I used his
methods, and so did some of my collaborators, to prepare
hundreds of slides that were stained by several classical
procedures and by immunohistochemical methods for seven
or eight neurotransmitter-related antigens in rodents'
myenteric plexuses. This was about 15 years ago, and all the
work has been published. If you think such methods might be
workable with vibratome sections of fixed brain, ask for
more help. Enteric nervous system whole-mounts are more
robust physically than sections of brain because the
fragile neural elements are sandwiched between layers that
contain collagen and smooth muscle. If you think your
sections of gelatin-embedded and postfixed CNS are tough
enough, ask for more information.
Another suggestion: Make the Subject Line of your email
more informative. If you put "Section adhesion" you will
attract more attention. The words "my problem" probably
induce nearly everyone to hit the delete key.
John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London, Canada N6A 5C1
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