Dear Beth,

Perhaps you could try the resin route - fix in OsO4 then process to
epoxy resin.

Fix (and stain) the lipid using 1% OsO4 then dehydrate the cultures in
the plate 50% to 100% alcohol. You have to avoid using link reagents
such as propylene oxide or xylol due its dissolving effect on the
plastic dish, so infiltrate the culture with 50:50 100% alcohol and
epon. Follow this with three x 30 min changes of pure epon at 50'C to
remove traces of alcohol. Apply fresh epon to just cover the bottom of
the petri dish, place level in a 60'C oven and allow to polymerise over
night.
The cultures can be examined at low mag through the epon and petri dish,
or the epon layer can be separated from the dish by prising off with a
scalpel blade. having broken or sawn off the edges of the petri dish.
The released epon and cell layer can be stuck onto a suitable stub for
enface sectioning, or embedded on edge for sectioning at right angles to
the plane of growth. Paragon staining of the epon sections gives an
enhanced stain of the lipid structures as well as a background
counterstain. The sample could then be prepared for ultra structural
examination if desired.
We have used this technique successfully for many years, and will be
happy to supply you with a detailed method and safety protocol if you
wish.

With best regards,

Alastair McKinnon
Dept of Pathology, EM & Resin Histology Unit
University of Aberdeen
Scotland, UK
a.d.mckinnon@abdn.ac.uk
tel 01224 552502
fax 01224 663002

Date: 19 Oct. 1999 09:15:40 -0500
From: HistoScientific <histosci@shentel.net>
Subject: Staining Cell Cultures

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.

Sincerely,

Beth Poole