Permanent aqueous mounts (was Re: Crystal mount)
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From: | "J. A. Kiernan" <jkiernan@julian.uwo.ca> (by way of histonet) |
To: | histonet <histonet@magicnet.net> |
Reply-To: | |
Content-Type: | text/plain; charset="us-ascii" |
This is quite long. Don't bother reading it if you never
need a mounting medium that mixes with water, or if you
are happy with the aqueous medium you currently use. These
notes are about optimizing the microscopy and keeping
the slides for many years.
On Fri, 11 Dec 1998, Katri Tuomala wrote:
> I used Crystal Mount for several years, when using AEC as chromogen. I
> found that it preserved the staining well except maybe turned AEC a bit
> brownish in time. I did also on occasion applied a coverslip on it with
> Permount to mainly achieve a better optical resolution for
> microphotography. It would not be cost effective to do on all slides.
> Crystal Mount itself is expensive, which was one of the reasons we
> eventually switched over to DAB. Katri:-)
This sounds like a good product if it can evaporate to dryness
and retain its optical and protective properties. I wonder if
any public-domain aqueous media can do this. The requirements
for such a medium are that it won't deposit crystals when it
evaporates and that it won't be incapaple of evaporating. The
second condition probably excludes anything containing
glycerol. The first is more difficult. A polymer such as gum,
polyvinylpyrollidone or gelatin might fill the bill,
especially in the presence of a sugar that can form a syrup
that's almost solid. Fructose (laevulose) has this reputation,
and is a good aqueous mountant. Very cheap, but for some
reason somewhat too acidic for basic dyes, but can be buffered.
It's not 100% predictable; I've had fructose crystallize under
coverslips, but you can quickly remove all the mess with
water and then try again.
An old and certain way to make a solid and perhaps permanent
aqueous mount is to use a smaller-than-usual coverslip and as
little aqueous mountant as possible. You now allow a few days for
water evaporation at the edges of the coverslip. If you don't the
refractive index will be too low; unstained parts of the section
will be visible. (Sometimes that's wanted,in which case just mount
in a minimal drop of glycerol under the small coverslip.) It's
important to keep the top of the coverslip completely free of
dust, condensed moisture and anything else insoluble in xylene.
When the aqueous mount with its little coverslip looks OK,
give it a swish in clean xylene to remove any grease and apply
a larger coverlip in the usual way, as if the preparation were
a very thick (200 micron) section. The aqueous mount is now
sealed into a resinous medium. The coverslip thickness is at
least 340 microns. This reduces the resolution of a
microscope objective without an adjustable "coverslip collar,"
and if the section itself is a thick one, its lower levels
may be beyond the working distance of anything cheaper than
a $20,000 water-immersion objective. This won't be a problem
unless you're counting mitochondria or peroxisomes, but the
unduly thick double coverslip can still be annoying because it
necessitates much fiddling with the fine focus and substage
aperture. These annoyances can be reduced. Read on.
A refinement of the above technique is especially valuable
for cryostat sections collected onto coverslips rather than
slides. You can use a small or a large coverslip to collect
the section. After staining, make the aqueous mount on a
larger or smaller coverslip. Wait for evaporation etc as
explained one paragraph up, and then do the resinous mounting,
with the larger coverslip uppermost and the smaller one
applied to the slide. Now you have only the thickness of one
coverslip (and an unknown but small depth of 1 mounting medium)
between the specimen and the front lens of the objective.
For dehydrogenase histochemistry (seeing mitochondria in unfixed
cells; usually in tissue whose general microanatomy suffers
quite a lot to get this result), collection of sections on
coverslips is a must. For lipid staining with Sudan dyes, it's
often OK to collect the sections on slides and have double
thickness coverslipping, because high resolution may not be
needed.
Returning to Crystal Mount. If it's expensive, consider the
alternatives mentioned above. If it's really good and you
need only small amounts, use it. If you are a research worker,
never use any product without finding out and learning about
its composition. The results of research are questionable if
any part of the methodology is not fully understood by the
investigator. This applies even to a mounting medium. How
certain can you be that it faithfully preserves the stain?
And for how long? And how do you know it's that good?
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
Phone: (519) 679-2111
FAX (Department): (519) 661-3936
E-mail: kiernan@uwo.ca
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