Dear Giulia,

it will be a little bit difficult to point you to "ONE" staining method for Myelin sheaths (guessing brain....or else??) in this short reply. There are plenty of original articles out in the wild which can be found by either PubMed and Google search as well (to name only two possibilities).

Just as a "starter" I shall add 2 articles as pdf attached, if you have further questions, please reply and ask more in detail .... Best wishes and good luck...Wolfgang

and the second one:

hopefully helpful for decision making...regards, Wolfgang

Hi Giulia Bonetto

If you have more un-processed but fixed tissues you can use osmium tetroxide for staining. The protocol is described in many publications such as :

Microsc Res Tech. 2008 Jul;71(7):497-502. doi: 10.1002/jemt.20577.

A simple protocol for paraffin-embedded myelin sheath staining with osmium tetroxide for light microscope observation.

Best regards

Dear Gaber, dear Giulia,

somehow deplorable that we haven't got any reply so far from Giulia (since this would be the initial point to help her basically). Naturally for myelin sheath localisation / demonstration histologically, use of Osmium tetroxide is recommended (which has been used for a long time mainly as a[n unbuffered] fixative for, as well as especially to demonstrate the myelin sheaths / myelin lamellae (Cf. PEASE 1964 and older reports before 1900, introduced for EM-studies by PALADE in 1952).

@ Gaber, your mentioning the DeScipio paper 2008 (cf: http://www.ncbi.nlm.nih.gov/pubmed/18320578; Abstract: http://onlinelibrary.wiley.com/doi/10.1002/jemt.20577/abstract or (pdf, if open access): http://onlinelibrary.wiley.com/doi/10.1002/jemt.20577/pdf ) is greatly acknowledged..... but it should be mentioned that the reported technique (which more / less is analog to the GA-OsO4-fixation schedules for EM-preparations) was applied to nerve fibre segments of about 10 mm length (their diameter has not been described but can assumed to be in the range of approx. up to 3-5 mm at the largest). This is in favor of the low penetration depth of OsO4 at RT, reported to be some 0.5-0.6 mm / h (worse at 4 degr. C, cf. Hayat 1989: first hour approx. 800µm, further exposure to OsO4 with only a strongly slowed down diffusion rate). So, if you have to examine e.g. bigger brain specimens (initially FA-fixed, postfixation with (2% buffered) OsO4 solution it might be necessary to limit the brain tissue slices' thickness to 1 .0 - (max) 1.5 mm for the OsO4 treatment (incubate with all safety recommendations for not longer than 2 h at RT).

Best wishes and also regards,

Wolfgang

In the cross section of peripheral nerve, why myelin sheath is stained black with osmium whereas the surrounding fat cells in epineurium doesn't stain in black?

Lipid globules in fat cells are dissolved by solvent chemicals during staining but not the myelin sheath?

Sorry for my poor understanding of principles in osmium tetroxide staining.

Thank you

Hi Gialia,

It depends upon your interest and the magnitude of demylination. Immune staining such as MBP or PLP might be better. LFB staining is commonly used. However, i prefer to use silver staining as you can see even fibre.

Thanks

Monokesh

Article A modified technique for high-resolution staining of myelin

Dear Wong Kah Hui , apologies for a really late answer to your request or thoughts 3 years ago... your reply went online at a time I was very busy and exhausted due to my retirement from my position as well as the EM-Lab which was my 'second wife' for nearly 35 years ....so now I found the ResearchGate-notification in my Outlook Message system .... but unedited...

If there is still demand for explanations, I would like to say the following (in brief...if you have further questions, please leave a message):

myelin is not only "fat" or "lipid" which is extracted due to processing as we do this routinely when we fix or process brain or nerve material chemically.

There is a difference not only in the fixation but also in the nature of the two components (myelin and the lipidic proteins vs. "fat" lipid in fat droplets or fat vacuole of fat cells in surrounding epineurial connective tissue).

I do not know whether you deal with "histochemical staining" on previously fixed, then paraffin embedded and finally deparaffinized sections for light microscopy (=Histology), or you comment on TEM-processing techniques and sections with their black (or only faint) staining of myelin vs. [predominantly] extracted fat vacuoles in resin section (semithin, 1µm 'thick' sections).

It is not quite easy to describe the circumstances and physical processes and imponderabilities you are complaining of... (see above, quote: 'Sorry for my poor understanding of principles in osmium tetroxide staining...' and distribute 'knowledge' in brief....

Therefore I would like to invite you, to visit other (re)sources on Researchgate,

like, e.g.,:

https://www.researchgate.net/post/Does_anyone_have_ideas_about_lipid_fixation_for_fat_staining_in_paraffin_sections?

where you will find - for sure - at least some answers (also try -and find an intriguing micrograph demonstrating that "fat cells/fat vacuoles" CAN BE BLACKENED / ARE STAINED by OsO4*)....) in thread:

*) provided you have used some smart approaches to decrease elution by chemical processing and increase mass contrast of OsO4-stain/binding to the biological substrata....

==> https://www.researchgate.net/post/Is_there_any_suitable_staining_method_protocol_for_lipids_in_paraffin_sections....

and - as a real measure and information - far from my knowledge- authored by a famous chemist, Prof. John KIERNAN, Canada, I enclose here an article he wrote 2007 about the matter "Lipid Histochemistry":

Also an original work on Lipid fixation by Tracy & Wally 2004 I recommend to read....

My best wishes and regards, good luck,

W.H.M.

NB: the reply was edited once some minute after initial posting for only minor spelling and grammatical errors.

PS: for the article mentioned by @Monokesh Kumer Sen [Re# 006] I would like to refer to:

https://www.researchgate.net/publication/7459208_A_modified_technique_for_high-resolution_staining_of_myelin

and my Comment [01] there....

Dear Prof Wolfgang H. Muss,

Its great to read your two articles on the myelin staining. But Can you shed some light on its quantification. We have done Weil myelin staining for stroke injured rat brains. We do have coronal sections. Do you have any method or protocol to map the mylinated regions and its quantification

Dear Prof. Dayalan Sampath , as -with your additional RG-message you wanted me to respond to your e-mail-address, I already did so... Hope you are satisfied.

Best wishes and regards, cordially yours,

Wolfgang