I am trying to observe autophagosome in the starved MEFs cell by TEM (600,000x), but I have never seen the double-membrane of autophagosomes. I only saw a lot of single-membrane vacuoles. Look like there are no any double-membrane autophagosome at all. I really appreciate If you could tell me how to treat cells, collect the cells and fix them to get better TEM image for double-membrane autophagosomes.
Please have a look at page 35f in the attached publication. Maybe it can give you some ideas how to improve your experimental setup.
Regards, Christian
Article Guidelines for the use and interpretation of assays for moni...
Dear JinYu Zhang,
In case you need personal help in interpretation of TEM images, please send some of your images to me (using my Helsinki e-mail).
Best regards,
Eeva-Liisa Eskelinen
Thank you, Eeva-Liisa. My images are all very cloudy, maybe because the resolution is not high enough. I'm still working on it. Hope I will find the clear photos and send them to you.
Dear JinYu Zhang,
an another source is Chapter 5 in the book entitled Methods in Enzymology, Vol 543pp. 89-114, where You can find sections 2.3. Guidelines for the correct identification of autophagic vacuoles by TEM.
Best regards,
Łukasz Mielańczyk
http://www.sciencedirect.com/science/article/pii/B9780128013298000052
x600,000 is a way too high magnification for stained biological specimens. Do not expect to get good image at so high magnifications. You can better use magnifications in 50,000-100,000 range.
I too am having issues seeing autophagic membranes (in Drosophila fat body cells) and already read all of the above-mentioned papers.
I don't think she is really asking how to identify the membranes, but how to improve membrane visualization. I don't know the details of your sample prep, but you can try adding potassium ferricyanide (~1.5%) to your OsO4.
I hope that helps!
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