@ Daniel & Ulf: A Good & Happy New Year ! (Apologize for the long reply…)

Dear colleagues, thank you for your comment on this (for me) really “mysterious” staining procedure “Stevenel’s Blue”.

I had some days of thinking over your vibrant postings in favor of using this staining method. But: I have to add some minor critical remarks for anybody who is going to use the method or would think of staining with Stevenel’s Blue might be promising. Since the matter also for me is quite relevant, the following excursion to a minor part is based on own knowledge and experience and for the most extent on (hopefully thoroughly) reviewing and studying relevant literature and some interesting web-postings found by googling.

“Long ago” I have used this staining method for staining of special preparations of (‘hyaline’ aged human costal) cartilage embedded in epoxy resin and for ONE of multiple fixation protocol(s) I got really exciting differential staining in BLUE, bluish AND RED-reddish color shades/tones in areas of so called “amianthoid degeneration”(see example 1982 enclosed as jpg).

A following localization study of the two general “colors” RED & BLUE, + several nuances in between [intensive, to pale shades] to stained structures by TEM revealed interesting structural properties of the differently stained areas.

Unfortunately at that time I was not able to reproduce that specific staining (by red & blue-tones) again for samples fixed by other fixation schedules.

The term: (at least as subject keyword as well as within the text of the relevant section “Thiazine”-) is not listed in CONN’s Biological Stains (10th Ed), A Handbook of Dyes, Stains and Fluorochromes for Use in Biology and Medicine, Ed by R.W. HOROBIN and J.A. KIERNAN; a Biological Stain Commission Publication; BIOS Scientific Publishers, Oxford, UK. 2002 (ISBN 1 859960995) and this has –undoubtedly – a reason:….“Stevenel’s Blue”, per definitionem product of an oxidation of “Methylene Blue” most probably gives rise to several dyes: i. e. (polychromed) methylene blue, Azure A, B, C, methylene violet Bernthsen, Toluidine Blue & others (keywords: Romanowsky stains: Giemsa’s, Leishman’s, Wright’s stains also are produced from methylene blue by oxidation). More exactly this might be found in:

--------------

[ Citation ]:“Conn's Biological stains, Lillie, RD, revised by Stotz EH and Emmel VM: H J Conns Biological Stains. pp 423-424, Ninth ed., Williams and Wilkins Co,

Baltimore MD 1977 has explanation of what happens to the methylene blue when

oxidized by KMNO4. The by- products of the methylene blue oxidation are toluidine blue, methylene violet, thionin, Azure A and other Azures along with residual methylene blue left in the solution.

[end citation from Gay Callis’ comment on Stevenel’s Blue at: http://lists.utsouthwestern.edu/pipermail/histonet/2009-December/048190.html ]

That posting also mentions (under the title) the possibility to purchase a commercially available staining solution called Sandersons Rapid Bone stain (which is said to be exactly the same solution as Stevenel’s).

>

Her recipe is proprietary and stains with the same results as the Maniatopoulos method. We did the comparison in our lab and had identical results. Because the Stevenel's is such a royal pain to make in the lab, I suggest buying the Sanderson Bone stain. It is money well spent to avoid a long day of stain preparation.[End of Citation]>>

(other related postings Sanderson’s rapid bone stain + Acid fuchsin cf. (John Kiernan, 2009) @

http://lists.utsouthwestern.edu/pipermail/histonet/2009-December/048198.html

http://lists.utsouthwestern.edu/pipermail/histonet/2009-December/048171.html,

by Jack Ratliff @

http://lists.utsouthwestern.edu/pipermail/histonet/2009-December/048176.html

http://lists.utsouthwestern.edu/pipermail/histonet/2009-December/048179.html &

Re by Jack Ratliff @

http://lists.utsouthwestern.edu/pipermail/histonet/2009-December/048184.html

------------

There are only very sparse original literature references for “Stevenel’s Blue” found by Google, PubMed [= 14 results], or other online search which mostly have been published in the 1980ies and 1990ies. A lot of them are only stating the use of Stevenel’s Blue stain (+ reference given).

The most common (and perhaps most cited) article for Stevenel’s Blue is:

Stevenel's Blue, an excellent stain for optical microscopical study of plastic embedded tissues.

by del Cerro M, Cogen J, del Cerro C in Microsc Acta. 1980 May;83 (2):117-21

(cf: http://www.ncbi.nlm.nih.gov/pubmed/6156384 )

Also – concerning critical reading of the Stevenel’s Blue stain – I would like to point you to the Histonet-post made in April 2003 by Gayle Callis @ http://www.histosearch.com/histonet/Apr03/Stevenelsblue.html. You’ll find really interesting points, especially if you try also to see all postings (cf. “previous message - next message)

An interesting (and most possibly very promising) “variant” of Stevenel’s Blue staining is:

Stevenel’s Blue and counterstained with Van Gieson’s Picrofuchsin (SVG stain) according to the methods of Maniatopoulos et al. (1986).

cf. >

For that variant there are some publications using that modification and going through several papers, I was not very convinced about the real outcome of proper Stevenel’s BLUE staining rather than displaying Van Gieson’s coloration shades…

Very impressive results with the latter modification one can find in an article by:

Dutta ROY T et al, Performance of hydroxyapatite bone repair scaffolds created via three-dimensional fabrication techniques

( J Biomed Mater Res A. 2003 Dec 15;67(4):1228-37

[cf: http://www.ncbi.nlm.nih.gov/pubmed/14624509 =

http://onlinelibrary.wiley.com/doi/10.1002/jbm.a.20034/abstract or =

http://onlinelibrary.wiley.com/doi/10.1002/jbm.a.20034/pdf , if you have online access)

From my view it would be at least mandatory to cite the source, C.I. and properties of the Methylene Blue used for making (your) Stevenel’s Blue. Otherwise it might be a spiky way to achieve successful, satisfying staining results, I guess.

So at the end of my excursion into theory and practice, I would like to ask you dear Daniel, as well as dear Ulf, honestly, whether you could send me or post any tip concerning your “optimized Stevenel’s Blue recipe (if available).

Thanking you in advance,

best regards,

Wolfgang MUSS, SALZBURG, AUSTRIA

When optimized, Stevenel's Blue provides a wider gradient of colors or shades that better highlight differences in the matrix and especially different cell types. For more information and opinions, I suggest posing this question in the NSH Hard Tissue Forum which you can find on Yahoo Groups. http://groups.yahoo.com/group/hardtissue

Stevenel's Blue nicely distinguishes osteoid formation, importantly Stevenel's Blue generates very photogenic specimens for publication.

@ Daniel & Ulf: A Good & Happy New Year ! (Apologize for the long reply…)

Dear colleagues, thank you for your comment on this (for me) really “mysterious” staining procedure “Stevenel’s Blue”.

I had some days of thinking over your vibrant postings in favor of using this staining method. But: I have to add some minor critical remarks for anybody who is going to use the method or would think of staining with Stevenel’s Blue might be promising. Since the matter also for me is quite relevant, the following excursion to a minor part is based on own knowledge and experience and for the most extent on (hopefully thoroughly) reviewing and studying relevant literature and some interesting web-postings found by googling.

“Long ago” I have used this staining method for staining of special preparations of (‘hyaline’ aged human costal) cartilage embedded in epoxy resin and for ONE of multiple fixation protocol(s) I got really exciting differential staining in BLUE, bluish AND RED-reddish color shades/tones in areas of so called “amianthoid degeneration”(see example 1982 enclosed as jpg).

A following localization study of the two general “colors” RED & BLUE, + several nuances in between [intensive, to pale shades] to stained structures by TEM revealed interesting structural properties of the differently stained areas.

Unfortunately at that time I was not able to reproduce that specific staining (by red & blue-tones) again for samples fixed by other fixation schedules.

The term: (at least as subject keyword as well as within the text of the relevant section “Thiazine”-) is not listed in CONN’s Biological Stains (10th Ed), A Handbook of Dyes, Stains and Fluorochromes for Use in Biology and Medicine, Ed by R.W. HOROBIN and J.A. KIERNAN; a Biological Stain Commission Publication; BIOS Scientific Publishers, Oxford, UK. 2002 (ISBN 1 859960995) and this has –undoubtedly – a reason:….“Stevenel’s Blue”, per definitionem product of an oxidation of “Methylene Blue” most probably gives rise to several dyes: i. e. (polychromed) methylene blue, Azure A, B, C, methylene violet Bernthsen, Toluidine Blue & others (keywords: Romanowsky stains: Giemsa’s, Leishman’s, Wright’s stains also are produced from methylene blue by oxidation). More exactly this might be found in:

--------------

[ Citation ]:“Conn's Biological stains, Lillie, RD, revised by Stotz EH and Emmel VM: H J Conns Biological Stains. pp 423-424, Ninth ed., Williams and Wilkins Co,

Baltimore MD 1977 has explanation of what happens to the methylene blue when

oxidized by KMNO4. The by- products of the methylene blue oxidation are toluidine blue, methylene violet, thionin, Azure A and other Azures along with residual methylene blue left in the solution.

[end citation from Gay Callis’ comment on Stevenel’s Blue at: http://lists.utsouthwestern.edu/pipermail/histonet/2009-December/048190.html ]

That posting also mentions (under the title) the possibility to purchase a commercially available staining solution called Sandersons Rapid Bone stain (which is said to be exactly the same solution as Stevenel’s).

>

Her recipe is proprietary and stains with the same results as the Maniatopoulos method. We did the comparison in our lab and had identical results. Because the Stevenel's is such a royal pain to make in the lab, I suggest buying the Sanderson Bone stain. It is money well spent to avoid a long day of stain preparation.[End of Citation]>>

(other related postings Sanderson’s rapid bone stain + Acid fuchsin cf. (John Kiernan, 2009) @

http://lists.utsouthwestern.edu/pipermail/histonet/2009-December/048198.html

http://lists.utsouthwestern.edu/pipermail/histonet/2009-December/048171.html,

by Jack Ratliff @

http://lists.utsouthwestern.edu/pipermail/histonet/2009-December/048176.html

http://lists.utsouthwestern.edu/pipermail/histonet/2009-December/048179.html &

Re by Jack Ratliff @

http://lists.utsouthwestern.edu/pipermail/histonet/2009-December/048184.html

------------

There are only very sparse original literature references for “Stevenel’s Blue” found by Google, PubMed [= 14 results], or other online search which mostly have been published in the 1980ies and 1990ies. A lot of them are only stating the use of Stevenel’s Blue stain (+ reference given).

The most common (and perhaps most cited) article for Stevenel’s Blue is:

Stevenel's Blue, an excellent stain for optical microscopical study of plastic embedded tissues.

by del Cerro M, Cogen J, del Cerro C in Microsc Acta. 1980 May;83 (2):117-21

(cf: http://www.ncbi.nlm.nih.gov/pubmed/6156384 )

Also – concerning critical reading of the Stevenel’s Blue stain – I would like to point you to the Histonet-post made in April 2003 by Gayle Callis @ http://www.histosearch.com/histonet/Apr03/Stevenelsblue.html. You’ll find really interesting points, especially if you try also to see all postings (cf. “previous message - next message)

An interesting (and most possibly very promising) “variant” of Stevenel’s Blue staining is:

Stevenel’s Blue and counterstained with Van Gieson’s Picrofuchsin (SVG stain) according to the methods of Maniatopoulos et al. (1986).

cf. >

For that variant there are some publications using that modification and going through several papers, I was not very convinced about the real outcome of proper Stevenel’s BLUE staining rather than displaying Van Gieson’s coloration shades…

Very impressive results with the latter modification one can find in an article by:

Dutta ROY T et al, Performance of hydroxyapatite bone repair scaffolds created via three-dimensional fabrication techniques

( J Biomed Mater Res A. 2003 Dec 15;67(4):1228-37

[cf: http://www.ncbi.nlm.nih.gov/pubmed/14624509 =

http://onlinelibrary.wiley.com/doi/10.1002/jbm.a.20034/abstract or =

http://onlinelibrary.wiley.com/doi/10.1002/jbm.a.20034/pdf , if you have online access)

From my view it would be at least mandatory to cite the source, C.I. and properties of the Methylene Blue used for making (your) Stevenel’s Blue. Otherwise it might be a spiky way to achieve successful, satisfying staining results, I guess.

So at the end of my excursion into theory and practice, I would like to ask you dear Daniel, as well as dear Ulf, honestly, whether you could send me or post any tip concerning your “optimized Stevenel’s Blue recipe (if available).

Thanking you in advance,

best regards,

Wolfgang MUSS, SALZBURG, AUSTRIA

Wolfgang,

That is an excellent review of the available information. Thank you. Interestingly, Jack Ratliff, whom you cite, worked for me a few years ago and that is where my experience with Stevenel's/Sanderson's began. Jack did a lot of work to optimize the staining procedure with and without SVG counterstain in both thick sections (Exakt cut and ground) and deplasticized thin sections. The SVG counterstain is very nice for some applications, but if my memory is correct, the intensity of the SVG was not reliable on thick ground sections and would sometimes obliterate many of the subtleties of the Stevenel's.

I now use the commercially available Sanderson's Rapid Bone Stain. It is quite reliable, but we have not used it much in the last year or two, and I only use it on thin sections these days. The optimization I was referring to was really in the procedure. I have found that we need to optimize the staining time and any differentiation steps depending on which tissue we wish to analyze. For example, increasing staining time and differentiation can highlight differences in bone matrix staining, but washout differences in marrow (including Ob and Oc) staining. In contrast, I prefer longer staining with less differentiation for cartilage, and less intense stain with little, but some, differentiation for Ob and Oc.

Jack is probably the best expert on this, especially for thick ground sections. I can put you in touch with him if you want.

Cheers

Dan

Dear Daniel, thank you for your thorougly and really appreciated reply. Yes there might be improvement by fiddling around the whole procedure....

If you don't mind, I would like to accept your kind offer to put me in touch with Jack Ratliffe... perhaps we can talk about the "mystery"...(:-)) a little bit!

(my e-mail at work: w.muss_at_salk.at )

Very best regards,

Wolfgang