Dear Gaber,
the advice of Hannes Ebner perhaps is a great and extensive one (all kinds of TEM-Methods included), but I guess the access to the article (Chapter 27 in Methods in Cell Biology; Volume 96, 2010, Pages 649–670
- Electron Microscopy of Model Systems-
cf. http://www.sciencedirect.com/science/article/pii/S0091679X10960275 )
will not be is not free of charges (PPV-View?)
(But perhaps you have gotten it by e-mailing from Hannes meanwhile and I wonder if it would be possible to get a pdf-version of that article from Hannes only for personal use??).
I personally have done only few cell culture monolayers with sufficient quality during my work in TEM, so it might be possible to add here a "simple" protocol (for "simple TEM-observation" without Cryo-options FF or HPF etc...) which is: adjust your (usual) fixative for mosmol & pH with regard to the cells you want to fix. Fixation time @ RT usually is "a tenth" of tissue specimen fixation time: 10-15 min may be sufficient. Wash well and process as usual (depending on the type of culture dish [ PP, Styrol?] CAVE solvent properties of acetone and propyleneoxide! during dehydration and infiltration): ascending alcohols (use ethanol, 5-10 min each step will be sufficient time), eventually use acetonitrile as the intermediate, latter also mixing with the resin (cave: hydrophilicity and watermiscibility of acetonitrile in tropic circumstances...). Besides other publications I have in my files another (web) more general advice: cf:
http://www.rndsystems.com/literature_fixation_ihc_icc_samples.aspx
- Fixation of Cultured Cells -
In contrast to tissue samples, cultured cells can be fixed for a shorter period of time with a less concentrated fixative solution. For example, fixation with a 2% formaldehyde solution for 20 minutes at room temperature is often sufficient to preserve both cellular morphology and antigenicity
- [personal remark: this for Imm-histological purposes; classical PLP-fixative for IHC and TEM, FA-GA-Fixative and eg. Acrolein-GA-fixative(naturally each solution/micture buffered accordingly, for classical TEM, lower concentration of aldehyde(s) are appropriate].-
Fixation of cultured cells is usually achieved by simply discarding the culture medium and adding the fixative solution. However, the change in surface tension following removal of the culture medium can damage some cell types. If this is the case, the fixative can be added directly into the culture medium. For example, adding the same volume of 4% formaldehyde as the volume of culture medium will result in a 2% formaldehyde solution, which is strong enough to pre-fix the cells. After 2 minutes, the pre-fixation culture medium should be replaced with a fresh volume of 2% fixative. The pre-fixation step makes the cells more rigid so they can withstand any potential deleterious effects created by changes in surface tension.
Note: Fixation can result in hydrophobic cross-linking of tissue proteins. The time, temperature, pH, and fixative used will determine the degree of cross-linking. Once the fixation protocol has been optimized, the same procedure should be used consistently.
Best wishes and regards,
Wolfgang
Dear Gaber,
the advice of Hannes Ebner perhaps is a great and extensive one (all kinds of TEM-Methods included), but I guess the access to the article (Chapter 27 in Methods in Cell Biology; Volume 96, 2010, Pages 649–670
- Electron Microscopy of Model Systems-
cf. http://www.sciencedirect.com/science/article/pii/S0091679X10960275 )
will not be is not free of charges (PPV-View?)
(But perhaps you have gotten it by e-mailing from Hannes meanwhile and I wonder if it would be possible to get a pdf-version of that article from Hannes only for personal use??).
I personally have done only few cell culture monolayers with sufficient quality during my work in TEM, so it might be possible to add here a "simple" protocol (for "simple TEM-observation" without Cryo-options FF or HPF etc...) which is: adjust your (usual) fixative for mosmol & pH with regard to the cells you want to fix. Fixation time @ RT usually is "a tenth" of tissue specimen fixation time: 10-15 min may be sufficient. Wash well and process as usual (depending on the type of culture dish [ PP, Styrol?] CAVE solvent properties of acetone and propyleneoxide! during dehydration and infiltration): ascending alcohols (use ethanol, 5-10 min each step will be sufficient time), eventually use acetonitrile as the intermediate, latter also mixing with the resin (cave: hydrophilicity and watermiscibility of acetonitrile in tropic circumstances...). Besides other publications I have in my files another (web) more general advice: cf:
http://www.rndsystems.com/literature_fixation_ihc_icc_samples.aspx
- Fixation of Cultured Cells -
In contrast to tissue samples, cultured cells can be fixed for a shorter period of time with a less concentrated fixative solution. For example, fixation with a 2% formaldehyde solution for 20 minutes at room temperature is often sufficient to preserve both cellular morphology and antigenicity
- [personal remark: this for Imm-histological purposes; classical PLP-fixative for IHC and TEM, FA-GA-Fixative and eg. Acrolein-GA-fixative(naturally each solution/micture buffered accordingly, for classical TEM, lower concentration of aldehyde(s) are appropriate].-
Fixation of cultured cells is usually achieved by simply discarding the culture medium and adding the fixative solution. However, the change in surface tension following removal of the culture medium can damage some cell types. If this is the case, the fixative can be added directly into the culture medium. For example, adding the same volume of 4% formaldehyde as the volume of culture medium will result in a 2% formaldehyde solution, which is strong enough to pre-fix the cells. After 2 minutes, the pre-fixation culture medium should be replaced with a fresh volume of 2% fixative. The pre-fixation step makes the cells more rigid so they can withstand any potential deleterious effects created by changes in surface tension.
Note: Fixation can result in hydrophobic cross-linking of tissue proteins. The time, temperature, pH, and fixative used will determine the degree of cross-linking. Once the fixation protocol has been optimized, the same procedure should be used consistently.
Best wishes and regards,
Wolfgang
Hi, Vladimir, yes - this might be an important additional question (because it has certainly also implications on specimen preparatiion. But I'm quite sure about Gabers' reply on this point very soon....(:-))
Best regards,
Wolfgang
Dears vladimir and Wolfgang,
I want to process cell monolayer for T.E.M
Thanks
For my simple protocol I use cultures grown in 25-well plates on 13 mm Thermanox coverslips (I have better success rate in detaching specimens from Thermanox). Cultures are processed directly in wells along usual TEM protocols (fixation, OsO4, alcohol dehydration) with somewhat shortened time per step. Do not use propylene oxide (will dissolve wells), do not mix resin with alcohol. Directly after dehydration 3 changes of pure resin. Then I cut specimens with hand saw (jewelers saw or saw with spiral blade) – beams with long axis parallel to well axis. To detach Thermanox I heat them on warm (below 100C) hot plate and just pull piece of coverslip off. Up to 4-6 specimens could be cut from the same well in case if detachment was not successful. Then I cut sections under shallow angle to the culture surface. Usual problem – successful detachment of Thermanox. Block surface could have areas with remnants of coverslip, which should be trimmed off.
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