Permanent aqueous mounts (was Re: Crystal mount)

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From:"J. A. Kiernan" <> (by way of histonet)
To:histonet <>
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  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

   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

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