> Morning Jaclynn,
> 
> 	Well, let's see.  What I know is that to preserve metachromasia -
> Toluidine blue is one of the thazine dyes that is known for metachromatic
> staining - one is admonished NOT to use ethanol - I use tertiary
> butanol(TBA) - to dehydrate/differentiate a section.  Of course, in
> semi-thin staining, most procedures do not suggest anything but rinsing
> with water followed by drying before mounting - if that is how you prepare
> the semi-thin for viewing.
> 
> 	I have never put ethanol in my Toluidine blue stains.  With the
> above in mind as well as the normal semi-thin staining protocols with
> which I am familiar, if I did add ethanol it would be to restrict/prevent
> metachromasia in the preparation.
> 
> 	Why aging?  I can't give an all-knowing response, but if the ethanol
> slows some coordination of the buffer with the dye, then that would be an
> explanation.  
> 
> 	I presume your recipe is something like this:
> 
> 		1g TolBlue
> 		1g Sodium borate
> 		30ml 100% ethanol
> 		70ml water(d.i.)
> 
> 	If so then we are on the same page.  Perhaps what you need to do is
> add the ethanol, if you continue to use it, sometime (hours?) after you
> make up the aqueous dye-buffer solution.
> 
> 	My recipe comes from a small Lab Manual by:   Giuseppe
> Millonig(1976), Laboratory Manual of Biologic electron Microscopy, Mario
> Saviolo - Editore, Vercelli C.P. 182, Italy.  [Not easily found these
> days!] 
> 
> 
> 	Method 3(Millonig Ref:  Richardson, K.C., Jarret, L, Finke,
> E.H.(1960), Embedding in epoxy resins for ultrathin sectioning in electron
> microscopy, Stain. tech., 35: 313)
> 		
> 		0.5g sodium borate
> 		50ml water
> 		
> 		0.5g Toluidine Blue [O] -  C.I.  52040) - You should check -
> Lillie, R.D.(Ed.)(1977), H.J. Conn's Biological Stains(9th Ed.), Williams
> and Wilkins, Baltimore - for information on contrast between old and new
> dyes with this name.
> 
> 		Above sequence as given in Millonig.
> 
> 		I often use it fresh, and I can't ever remember a problem,
> even though, like you, I tend to make it in advance of need, at least by a
> day.
> 
> Hope this helps,
> 
> Fred Monson
> 
> Frederick C. Monson, PhD   
> Center for Advanced Scientific Imaging
> Schmucker II Science Center
> West Chester University
> South Church Street and Rosedale
> West Chester, Pennsylvania, USA, 19383
> Phone:  610-738-0437
> FAX:  610-738-0437
> fmonson@wcupa.edu
> CASI URL:  http://darwin.wcupa.edu/casi/
> WCUPA URL:  http://www.wcupa.edu/
> Visitors URL:  http://www.wcupa.edu/_visitors/
> 
> 
> ----------
> From: 	Jaclynn Lett
> Sent: 	Wednesday, May 22, 2002 11:32 AM
> To: 	Microscopy Listserver (E-mail)
> Subject: 	Toluidine Blue question
> 
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> 
> 
> Regarding a Toluidine Blue stain solution:
> 
> 1% Toluidine Blue O
> 1% Sodium Tetraborate Dodecahydrate
> 30% Ethanol
> in DDI
> 
> I've been using this stain for years on Epon-Araldite sections and
> Durcupan
> sections with no problems in getting satisfactory staining.  When I have
> 30-50mL remaining, I always make a new batch so it's ready the next time I
> need it , but this time I forgot.  I made a new solution and had to use it
> right away.  I stained my sections as usual, but the color of the sections
> was bright blue instead of the more purple-blue we desire.  Thinking that
> I'd made a mistake in preparing the solution, I made another batch and
> decided to wait overnight to try it, but again the sections were bright
> blue.
> 
> I was never aware of the need to do this, but does this solution need to
> "ripen" for a week or so before using?  I can't think of any other reason.
> The quality of our water hasn't changed, and everything else is the same
> as
> I've always used.
> 
> Thank you for any help you can give me.
> 
> Jaclynn M. Lett, Research Technician
> Electron Microscopy Core Facility
> Fay and Carl Simons Center for Biology of Hearing and Deafness
> Central Institute for the Deaf
> 4560 Clayton Ave.
> St. Louis, MO 63110
> 
> jlett@cid.wustl.edu
> 314-977-0257
> 
> 
>