7/22/97
Thank you to those of you who replied regarding LM mounting medium. It
appears as if oxidants in the medium are the main culprits. I am listing
these replies below.
One point worth mentioning about the common use of epoxy resins as a
coverslip mountant is that its refractive index is not 1.5 (I'm not sure of
its exact value). One may be able to get away with it working at low
numerical apertures, but it will take its toll at 0.65 and above.
Thanks again!
James Wesley-Smith
EM Unit
University of Natal
Durban, South Africa
I learned a trick from a DuPont-Sorvall rep (that tells you how long ago
that was!) that retards or eliminates ALL fading caused by oxidants in the
mounting medium. Add 1-2% BHT (the preservative used in bologna, hot dogs,
etc.) to any mounting medium. @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@
I have been using ENTELLAN which has ND20 1.49-1.5 and gives
excellent preservation of toluidine blue stained plant tissue for at least
4 years. I get it from Electron Microscopy Sciences who have a good list
of mounting media with refractive indicies in their catalogue.
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Hello
fading is the problem. We use these toluidine blue sections for histology
courses and we haven't find yet the no-fade mounting medium. Our best
choice is DEPEX, manufactured by GURR. Avoid EUKITT, fading occurrs in a
matter of hours. Some collegues have used cured epon, but it is a time
consuming process and fading does occur.
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I think the fading is caused by oxygen (which very
easily diffuses through hydrophobic media like resins).
We routinely leave such preparations uncovered, and add a drop of immersion
oil and coverslip to photograph. This prep is easily soaked off in xylene
to restain if necessary.
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I don't know about the refractive index, but I've used Epoxy resin as a
mountant quite successfully. It doesn't seem to fade Toluidine Blue
semi-thins.
DePeX is not too bad but I have had some fading over long periods.
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I have been working with plant material embedded in Spurr resin for many,
many years. One micron sections were stained with toulidin blue or other
specific stains and permanent mounted with Permount, Fisher Scientific, and
no fading for decades. I have not seen any fading using DePeX, but Spurr
resin sections usually get very wrinkeled.
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We use the following protocol for toluidine blue (TB) staining
and permanent mounting:
Frozen or dewaxed abd hydrated paraffin sections
0.1% TB in acetate or phosphate buffer (generally at pH 2-3 for
sulfated glycosaminoglycans) 5 min
Rinsing in buffer
Precipitation with a 6:1 mixture of 2% aqueous KI and Kferricyanide
2-3 min
Mount with a drop of 25% aqueous gum arabic containing 2% fructose
without coverslip! Form a layer of te mounting medium using a
glass rod. Let the gum arabic layer dry at room temperature in
horizontal position (it takes generally one night). The
refractive index of the dried gum arabic is practically
identical to that of the glass.
Mount in DPX or Canada using a coverslip.
This procedure is good to produce permanent metachromatic staining.
Dimethylmethylene blue (DMMB) is a better metachromatic dye (Aldrich
Co, or SERVA)
The protocol is similar, except the poststaining stabilization. For
this purpose, 2% aqueous ammoniummolybdenate is used.
DMMB is a very strong metachromatic staining. It is very useful for
mast cells, cartilage, sulfomucins. In many cases, we use it
successfully in 0.05 or 0.01% aqueous solution for 5-10 min.
For more inforations, see Modis, L.: Organization os the
Extracellular Matrix: A Polarization Microscopic Approach. CRC Press,
Boca Raton, 1991. Chapter 12.