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Dear Confocalists (and Susan and Guenter),

I am posting this answer to an off-list email in case anyone else is
interested, and to get it into the archives.

To seal coverslips, I have been using a substance called "Entellan"
(Merck, obtained through  EM sciences catalog) to seal coverslips. It is
marketed as a rapid mounting medium for Electron Microscopy, but it seems
to be good for sealing coverslips as well, because it hardens so fast.
Maybe this would be better than nailpolish.  This substance was
recommended to me by Ian Gibbins.

Also, I heard by word of mouth that nail polish is very detrimental to GFP
fluorescence, and I imagine this might be true of other fluorophores.

Vectashield has not worked for me because of tissue compression by the
objective lens during imaging, i.e. there is drift of the structures being
imaged because the pressure of the lens causes the mount to compress.  But
I am imaging very small structures in 3D, located in 150-micron tissue
sections.  The drift is a problem because the 3D structure is distorted.
But maybe this would not be a problem for other applications,
particularly with cell monolayers.

I have tried to use 'spacers', either 150 micron steel strips, or pieces
of coverglass cut with a diamond pencil, or simply scotch tape - all
without luck.  In the end I have found it best to use "Fluorsave" medium
from Calbiochem: this medium hardens in about 1 hour, after which I seal
the coverslip with Entellan.  I don't see any great effect on the tissue,
except maybe after about a week or so it does begin to pull on the tissue
a bit: probably because it continues to harden and polymerize during that
time.

If the specimen is sufficiently thin that drift is not a problem, the most
ideal mounting medium is probably Glycerol, due to its high refractive
index, which is closer to that of immersion oil.  Vectashield is quite
close to Glycerol in r.i., and in addition has some antifade ingredients.
But there are dyes that cannot be used with glycerol: all lipophilic dyes,
e.g. DiI and others of that kind.  These dyes will diffuse into the
glycerol and the image will be degraded.  Vectashield probably contains
gylcerol, so probably the same will occur with Vectashield.



On Mon, 20 May 2002, Susan Krueger wrote:

> Hello, Wes,
>
> I need to keep track of the current info on mounting media. I have very
> thick whole mounts, up to 250um, and am scanning with a large working
> distance, high NA, oil immersion, 100x objective, using the 488 line. Do
> you know of any more current info than the attached message? I searched
> the confocal archives for this. I do have a print-out of your table of
> aqueous mounting media.
> Unless you know of something better, I consider my best options to be:
> 1). Vectashield, with the coverslip sealed by epoxy or nail polish
> 2). Fluoromount, from Serva
>
> I would like to avoid a media that shrinks the tissue. The MOST
> important factor to me is come as close as possible to a refractive
> index match. We will eventually be deconvolving the images with
> Bitplane's Huygens software.
>
> Thanks,
>
> Susan Krueger
> Center for Computational Biology
> MSU
> Bozeman, MT
>


Hi Cynthia,

Since several years I mainly use Vectashield (Vector Labs.), which may be
diluted with glycerol 1:1. DABCO (up to 2%) can also be added to this mix,
but seems to interfere with the Hoechst dyes. Vectashield, on the other
side, seems not to be compatible with Texas Red, but caused no problems
with FITC, GFP, TRITC and the Cy dyes. Check any combination first!

Use high quality cover slips (e.g. 0.17 plus/minus 0.01 mm thickness) in
combination with lenses corrected for cover slips. Try to match the
refractive index of the mounting medium and the lens immersion medium. The
media mentioned above (refractive index of about 1.45) are suited better
than buffer-based media (about 1.35, e.g. PBS alone with DABCO etc.) when
using oil immersion lenses.

These mixtures stay liquid, so the samples have to be sealed. Preparations
in our hands stored at -20 C seem to be stable for weeks and even for
months and can repeatedly be taken out of the freezer (be cautious with
interpretations based on such old samples, but they still can be used for
demonstration purposes).

Preparations mounted in liquid media (and your objects embedded within!)
seem not to shrink in volume over time when sealed properly. Shrinking is
at least less than in embedding media which harden by drying (like Mowiol,
or ProLong from Molecular Probes).

To seal and to fix cover slips (separated from the slide by a thin layer
of
plastic cut off a freezer bag), I use nail polish or nail hardener, but
sometimes I run into problems with its bad long-time contact with the
slide
surface. (Slides may be "contaminated" with embedding medium, or some
mounting medium may penetrate slowly into or below the "hardened" glue.)

To any reader of this mail: Is there any information on the basic
composition of (acrylic?) nail polish or nail hardener? Or further
suggestions on fixing cover slips?


Best wishes,

Guenter


----------------------------------
Dr. Guenter Giese
MPI fuer Medizinische Forschung
Jahnstr. 29
D-69120 Heidelberg
Phone (Germany or 0-)6221-486-320
Fax   (Germany or 0-)6221-486-325
e-mail: [log in to unmask]
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