Re: Staining Cell Cultures

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From:Paul Klosen <klosen@neurochem.u-strasbg.fr> (by way of histonet)
To:histonet@magicnet.net
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Date:Wed, 20 Oct 1999 03:46:46 -0400
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A 10:07 19/10/1999 -0400, vous avez crit :
>Dear Netters,
>
>We have a client that would like to see lipid vesicles in cell cultures
>that he has developed in a Petri dish.  How do we get the cell culture
>on the microscope slide?  Is the culture something you can frozen
>section, then stain?  We work with the actual tissue most of the time,
>therefore we are not very experienced with cell cultures!  Any
>suggestions would be most appreciated.

Hi Beth,

Cell cultures are normally stained in the dish. Alternatively, you can try
to grow the cells on glass coverslips, which allow you to to mount the
cells then face down on a glass slide. Finally, a few manufacturers cell
specially designed culture dishes, that are plastic slides, with a wall and
a lid creating a culture dish. After fixing the cultures, you can get rid
of the wall and remain with a slide that you can then stain.

The problem with the coverslips and the special culture "slides", is that
often you establish special culture conditions to obtain a certain
differentiation of your cells. one of the conditions is the culture
substrate (either protein or a specially treated culture dish). If you
change to coverslips, you may not be able to recreate the same conditions,
and thus not be able to obtain the same differentiation.

As far as the staining of lipid vesicles goes, these little problems are
actually without relevance. One of the standard stains for lipid vesicles
in cell culture is Oil Red  (or Sudan Black, check either Culling's
textbook, or M. Gabe's textbook, if you read French). These are lipophilic
dyes that partition into the lipid vesicles. But then you cannot dehydrate
without losing your dye. So you end up using an aqueous mountant. Simply
use the Oil Red or Sudan Black procedure in the culture dish, counterstain
with Hematoxylin, mount in whatever aqueous mountant you use and put a
coverslip onto the cells in the dish (avoid Aquamount !!! this one tends to
diffuse the lipophilic dyes). Depending on the culture dishes and their
size, you may have to use an inverted microscope to observe the cells.

Paul Klosen
                                    -=-
                                   (o -) O
==========================3D=====oOo==(_)==OOo=============3D=====================
Paul Klosen, PhD
CNRS UMR 7518 Neurobiologie des Fonctions Rythmiques et Saisonnieres
Universite Louis Pasteur  12, rue de l'Universite
F-67000 Strasbourg, FRANCE
Tel. 03.88.35.85.04  Fax. 03.88.24.04.61
========================klosen@neurochem.u-strasbg.fr==================3D=======





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