[Histonet] Electron Microscopy protocols

[idimitro <@t> mun.ca]

Hi Joel, 

Lipids are fixed only by OsO4, so you need to use it as a post-fixative.
As a fixative in our lab we use Karnovsky fixative, then we use osmium tetroxide as a post-fixative/fixative for the lipids for 15 min. Then we wash in Na-cacodylate buffer for 5 min, and then dehydrate, starting with two changes of 70% ethanol ( for 10min.) and then we go to two changes of 95% and then absolute ethanol, followed by two changes of abs. acetone, then 50:50 acetone:resin and 2 changes of resin, then embedding and polymerization at the right temperature.

If your lipids are fixed properly they will be round droplets with characteristic appearance. OsO4 will blacken the tissue.
We always have excellent results with this protocol.

Hope that was helpful,

Iliana dimitrova,
Memorial university of Newfoundland,
Canada

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Joel Haas
Sent: June-05-13 3:00 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Electron Microscopy protocols

Hello all,
    I am working on developing an electron microscopy protocol for looking at lipid droplets in cultured cells. We have used thin section TEM previously and found that the morphology of the droplets is often deformed (should be spherical, shows up as egg shaped or lumpy). If possible we would like to avoid these types of preservation artifacts. Does anyone have suggestions for adapting the protocol to better preserve these types of structures--a triglyceride lipid core surrounded by a phospholipid monolayer?

Thanks in advance,
Joel
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