Hello Neha,  Toluidine Blue is as other basic dyes like cresylviolett or thionine not suitable for Axon stain. This dyes stain nuclei and Nissl-substance but no fibres. If the axons are myelinatede you can combine cresyviolett with luxol-fast blue (Kluever-Barrera-stain). But the best way to stain axons in paraffin sections is to use siver-impregnations like Bielschowsky's or Himes's silverstain  for axons. The methods are discribed in J.A. Kiernan: Histological & Histochemical Methods. Good luck!

Hi

Also, you can see this book:

Article Theory and practice of histological techniques / edited by J...

the best way ti stain them is osmiium tetroxyde which stains black axons and myelin, the only problem is a neurotoxin so it is very dangerous to use even at a 2 p/cent concentration. if you decide to use it please read the health and safety warnings very very carefully

Hi all! 

Thank you for your suggestions. As for the Osmium Tetroxide method, it seems this step is done after fixation and before paraffin embedding. The samples I have already been embedded in paraffin and sliced and mounted on slides. Will the osmium tetroxide still work if I incubate the already-made slides in it? 

Hi, i have never done it in this way, but as far as i know you should be able to do so, osmium will attach to fatty tissue, this will not change after paraffin treatment, my protocol was after fixing 10 min in osmium then wash and mount in resin, so the fixing part will work, as i told you i never did it in that way but if you have samples to spare and decide for osmium have a go and see if it works

Hello Neha,  Toluidine Blue is as other basic dyes like cresylviolett or thionine not suitable for Axon stain. This dyes stain nuclei and Nissl-substance but no fibres. If the axons are myelinatede you can combine cresyviolett with luxol-fast blue (Kluever-Barrera-stain). But the best way to stain axons in paraffin sections is to use siver-impregnations like Bielschowsky's or Himes's silverstain  for axons. The methods are discribed in J.A. Kiernan: Histological & Histochemical Methods. Good luck!

I performed such staining on semithin sections only and the results were very nice.

Regards,

Hanna Gendek-Kubiak

Hi all, thank you again for your responses, I've realized toluidine is not suitable at all and resorted back to silver staining!

Ihave sent my answer, but I cannot see it.So, I repeat: My biopsies were fixed with osmium, so together with toluidine blue it was very nice, because the myelin was impregnated with osmium and whole axons were very well visible.

Dear (Prof....Dr....) Hanna Gendek-Kubiak , would you mind telling us which 'specimens' you dealt with.... it might be interesting to know about "which" nerve-/axon-paths you and - in a "vertebral artery"- Neha Iftikhar has / have been talking. At the moment we do not know whether Dr. Iftikhar will read these answers (since the question was asked long ago) or if he/she meanwhile might have overcome the problem or doesn't need a solution of the staining problem depicted. Also I would be interested in the processing protocol (starting from the biopsy tissue itself, primary fixation, the fixative etc. but especially which resin and how you performed the TOLBLUE staining on your sections....) thanking you in advance, best regards and wishes, stay safe and healthy...WHM

Dear Professor Wolfgang H Muss,

I performed my stainings many years ago on guinea pig skeletal muscles using procedure for elecron microscope: glutaraldehyde, osmium tetroxide and Durcupan ACM resin. My toluidine blue staining was performed on semithin sections for light microscope.

Thank you for your wishes. I wonder why in Germany there is smaller percent of death in epidemia, than in other countries?

Regards and best wishes in this difficult time,

Hanna Gendek-Kubiak

Dear Hanna Gendek-Kubiak (hopefully it is no problem to omit the title /your titles here, otherwise: apologies!) , I am no "Professor", only retired private person....(:-)). Thank you for your specifications given in your recent answer. Interesting for me to be "kicked back" into my former profession and daily work....I used to perform processing and examination of clinical biopsies (children, adults) by TEM too, preceeded by light microscopical evaluation of semithin sections. I was 'tired of Toluidine blue staining' some years ago (1994,1995. cf.: enclosed pdf's - apologies for poor quality of the copy for pt1(1994) which I was able to find in my digital library / e-works-collection, ..and 2 images) and was able to create an improved polychromatic staining for semithin sections of resin (epoxide / Epon / Glycidether100) embedded tissues (predominantly muscle, skin, but also cornea, etc.). The recipe/protocol given in part1(Humphrey and Pittman, 1974) might work also on DURCUPAN ACM gorgeously !

Concerning your other question concerning Germany's low numbers of death due to Covid-19-infection: I don't know but (using e.g. Google translation if necessary) you might find an answer in the following blog provided bny XING today at:

http://www.xing-news.com/reader/news/articles/3060860?link_position=digest&newsletter_id=58737&toolbar=true&xng_share_origin=email

(if you can't find or open the blog please tell me and perhaps I should be able to send you a copied&pasted version (in a word.docx or pdf).

My best regards and wishes, stay safe and well... take care of you and yours, Wolfgang

Dear Wolfgang Muss,

Thank you very much for your answer and interesting files concerning semithin sections and staining of them. This protocol with fuchsin I tried, too. I think, your micrographs are very nice. I am surprised to see, that you worked with the same tissues (muscles, skin), as me. Now, I am retired, too, and perform some didactic duties in Histology and Embriology with medical students.

I think, my statement about staining of neurofibers with toluidine blue was not very precise...

Concerning coronavirus - I don't understant German at all, but I think that meaning of this text is something about level of health care?

Best regards,

Hanna

Dear @Hanna Gendek-Kubiak , thank you for your kind reply...since I was out to provide some dairy goods toand for the 24h nurse of my mother (99+ y.o.a) I only now can answer as short as possible...

As of your answer "... I think, my statement about staining of neurofibers with toluidine blue was not very precise... ..." believe me: IMHO answers always depend on a precise question.... as it partly was not the case with the initial question...

With regard to Toluidine blue it has been said that Tol-blue is no fiber - / fibre - (=axon-) stain as other single basic dyes would not stain axons too... (at least for resin embedded semithin sections viewed at HR-LM it might be possible that the small cross-section-area-dimensions - as is the case even in properly processed and sectioned histological sections - add to such an "invisible state").

IMHO another question to answer or to inform of would have been the expected axon "diameter" to be seen... (there are very small axons in the adventitia as well as such can be found nearly joining the BM of the endothelial cells,

which might be localized much better or more precisely by ancillary IHC-staining techniques

(as can be found in one of the following articles - not only! - too: Article Silver Acetate Autometallography: An Alternative Enhancement... ; Article Distribution of two VIP-related peptides, helospectin and pi... ; or Article Neuropeptide in der menschlichen Nasenschleimhaut [in German... respectively )

Concerning "Corona-number of deaths in Germany":

For your convenience I attach the offered XING.pdf (German translated top Polish -not knowing how effective Google did the automated translation...) My best wishes to you and yours, stay safe and well, Wolfgang