Dear all,
A lab asked to our electron microscopy facility to analyze whether antidepressants change autophagy in mouse hippocampus neurons with transmission electron microscopy. In order to do this, I will really appreciate if someone could provide me a detailed protocol for fixing mouse brains. This lab will do the experiments, fix the brains and send them to us (from Germany to Italy). How should be the better way to send the samples (in fixative, in buffer, etc.)? Moreover, about post fixation, should I use just osmium or osmium reduced potassium ferrocyanide?
Thank you very much!!!
Francesco
How should be the better way to send the samples (in fixative, in buffer, etc.) . In Fixative.
Osmium reduced and in buffer.
Place tissues in 4% paraformaldehyde for no longer than 48 hours at 4oC. The solution should completely cover the tissue. After 24-48 hours, tissue can then be stored in 1X PBS at 4oC for up to two weeks. Tissues may also be stored in 70% ethanol at 4oC.
Have a look at : Mikula, S., Binding, J. & Denk, W. Staining and embedding the whole mouse brain for electron microscopy. Nat. Methods 9, 1198–1201 (2012).
Behnam
Do not know about brain, but for other tissues I was getting better results after perfusion.
@Behnam Lak and other interested readers: (only for convenience):
Mikula, S., Binding, J. & Denk, W. Staining and embedding the whole mouse brain for electron microscopy. Nat. Methods 9, 1198–1201 (2012): the reference, its abstract on the Journal's webpage::
http://www.nature.com/nmeth/journal/v9/n12/abs/nmeth.2213.html
The article, on RG: https://www.researchgate.net/profile/Shawn_Mikula/publication/232532108_Staining_and_embedding_the_whole_mouse_brain_for_electron_microscopy/
as well as the supplementary information/Material: http://www.nature.com/nmeth/journal/v9/n12/extref/nmeth.2213-S1.pdf
You can find therein 'Perfusion fixation' of whole brain essential (there preferably ONLY with sodium cacodylate buffered ACROLEIN, instead of using buffered FA and GA or FA or GA-solution , Postfixation also very special, since in Acrolein!, the use of special OsO4 solution, the use of OTO (Osmium tet5roxide- Periodic Acid - Thiocarbohydrazide - Osmium Tetroxide) etc,.etc.
Not only I DO NOT recommend storage / delivery of fixed brain parts in 1x PBS, since PBS no time was and is a "TEM-coherent" buffer ....it is either suited (= pH- as well as tonicity-/molarity controlled) Na-Cacodylate.....or Na-Na-PO4 buffer systems....
'1x PBS' is hyoposmolar (0.01M!)
Will come back later on...
NB:edited after some 3 hrs for wrong word in place: "Postfixation also very special, since [ in Acrolein!] use of special OsO4-solution...
Article Staining and embedding the whole mouse brain for electron microscopy
My [Re#05 continued]:
concerning PBS: the "dilemma" of often equating 'PBS' ("phosphate buffered saline" ) with suited and also well defined 'buffer solutions' which commonly are used in (S, T-EM)-specimen Preparation can bve found also in the Q&A archive on RG: (e.g. https://www.researchgate.net/post/Does_anyone_know_the_procedure_to_prepare_a_01M_solution_of_PBS2?) you only have to "search" RG in "Questions" for " PBS phosphate buffered saline " or any other suited search phrase / keyword.
Last but not least:
IMHO it is not quite effective help to state something of real importance just as a 'substantive' (e. g.: "In Fixative..."). My experience was and honestly I would do it again: think about the task ,the goal of examination and which structures are to be localized (ALL, only neurons, axons, myelin sheaths, "tracts", only overall "histo-morphology....etc. etc.). Think twice.... (perhaps you forgot some crucial detail...) then decide, and follow critical SOP's. Then also you have / one has to decide to which point the specimen preparation has to be done by the experimenting institution, unless you are able to conduct safe transfer of the the specimens to the EM-Lab doing the final steps of processing (embedding, sectioning, staining, examining, taking images, etc., etc.). Also think about the modality: surface snail mail - parcel mail ('Post-parcel', EMS, UPC or other services), air mail....(nota: special conditions of transport could apply if you use fixative solution as transport medium). Regarding that last argument, it might be better to organize a most rapid transportation/transfer in suited buffer solution (regardless of whether this is commercially bought or [recommended] a buffer solution you have made/mixed up by yourself (since you could be sure you meet the right parameters needed....) Good Luck and my best wishes ! .....remember always: "garbage in - garbage out....." regardless of how expensive your total preparation and handling/transfer-transportation costs were.... Francesco: you are welcome....(thanking you for Rec.....(:-)) W.
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