tissue tek for mounting cells on coverslips?

Henk Veldman H.Veldman-2 at neuro.azu.nl
Mon Feb 25 07:24:31 EST 2002



Wolfgang Schechinger schreef:

> Dear all,
>
> Did anyone yet use Tissue Tek embedding glue for mounting cells grown on
> coverslips for fluorescence microscopy?
>
> Since people normally use it for microsections of tissue, I better ask here
> before I waste my samples. Actually, I planned to use PBS : glycerol 1:1, but
> accidentally, I have just discovered a bottle of TT and if it really glues, so
> I'd give my preference to it.
>

Well, PBS : glycerol 1:1 is not the best mountant for immunofluorescence.

A good mounting medium should protect against bleaching, have a high refractive
index and should preferably be solid or semi-solid.
A higher pH (~9) protects FITC against fading, so use Veronal-buffered saline
instead of PBS. Increasing the glycerol content to 90% will increase the
refractive index and the "thickness" of the solution. Adding an antifading agent
will protect your fluorescence against bleaching: p-phenylenediamine (1 mg/ml) is
very effective, but will turn a nasty brown after some time; DABCO
(1,4-Diazabicyclo-[2.2.2]-octaan, up to 10%) is more stable and almost equally
effective.
Use something like nail varnish or 20% polystyrene in xylene to seal your cover
slips around the edges and your preparations can be stored with such a mountant in
the freezer for months.

As for Tissue-Tek: I have never seen it used as a mountant. The main constituant
is polyvinyl alcohol (PVA), which has also been used as a component (at 10 - 15%)
in some semi-solid mountants for fluorescence. I'm not sure about the other
chemicals in Tissue-Tek, but you should always check things like embedding and
mounting media, plastic cover slips or immersion oil for autofluorescence and (if
applicable) transparency for UV.

Good mounting media are also commercially available from the larger immunoreagent
companies (for instance VECTORSHIELD from Vector).

Hope this helps. Good luck.

Henk

--
H. Veldman
Laboratory for Experimental Neurology (NMZ)
University Medical Center Utrecht (AZU)





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