We are trying to elucidate if we need fixation in order to perform TEM of a sample of liposomes. Do you think it's fundamental? Or can we skip that step and perform the uranyl staining of the suspension of liposomes. Thanks.
The samples must be monodispersed and as thinly as possible on the carbon film coating TEM grids. The small amount of the material is suspended in a solvent (slightly turbid solution). For this purpose,water/ethanol may be used but we use ethanol preferably because it dries quickly. The sample should be loaded in advance on TEM grid and allowed to dry overnight. Then put this sample for TEM analysis.
Regards
I would not consider uranyl the best negative stain for lissome in TEM. Bare in mind that the uranyl solution is acidic and your lissome might be damage during the staining/drying due to the low pH. If you could not see good clear membrane layers then try tungstate as it buffers the solution at pH around 7.4.
Thanks a lot. Some questions, does ethanol disrupt liposomes? We have the liposomes in a PBS buffer pH=5.8. I only have uranyl, not tungstate, but I will keep in mind the pH situation. Our liposomes are in a slightly acidic medium. Do we need to perform some kind of fixation before staining?
You may put the liposome solution on the grid then dry it simply by leaving it near heat source. DON'T place it on hot surface. You can carry the grid and make it face ( from a distance) a lamp for few seconds. Ethanol may and may not disturb liposome depending on your lipid composition, some liposomes already are prepared in ethanol.
Hi David,
See attached articles. Also:
Transmission electron microscopy imaging:
Liposomes prepared by using 100 nm polycarbonate filters
were studied under transmission electron microscope (TEM).
For this, the standard osmic acid method of Lewis and Knight
(1977) was employed. Five microliters of 1% (w/v) OsO4 was
added to 2 ml of the liposomal suspension which was then hand
shaken and centrifuged at 4 8 C, 100,000 g , for 60 min in a
Beckman L-80 refrigerated ultracentrifuge (USA). The supernatant
was discarded and the pellet was suspended in 2 ml PBS
(pH 7.4). Liposomes were then embedded in Taab TO28 epoxy
resin (Taab Embedding Materials, UK). The resin was
polymerised in an oven at 60 8 C for 24 h and ultra-thin pale
gold sections (50–90 nm) were obtained on a Reichet-Jung
ultramicrotome (Wien, Austria) with triangular knives prepared
on an LKB knife maker using a 5 mm 25 mm plate glass
knife.
The sections were examined using a JEOL JEM-1200 EX
transmission electron microscope (Japan) operating at 80.0 kV.
The TEM has its calibration checked every 6 months using
a standard graticule of 2160 lines/mm.
Hola David.
Como estas? Quisiera saber si lograste ver tus liposomas y como lo hiciste. Estoy enviando unas muestras a servicio técnico en la udea y no han logrado ver nada. Las tiñeron con acetato de uranio, adjunto las imágenes.
Te agradezco de antemano
David E. Bautista-Erazo You should definitely try 1% Gadolinium acetate. Works like wonder, just need to optimize conditions for washing time and staining time.
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