Re: toluidine again

From:Geoff McAuliffe

    Depends on the type of sections. You only need the tetraborate (which makes
the stain very alkaline) if you are trying to stain through epoxy (Epon,
Araldite, Spurr) sections. If you want metachromasia (varing shades of
blue/purple/red), you must have an acidic solution. If you are staining wax or
frozen sections, eliminate the tetraborate, eliminate the alcohol, and cut the
stain concentration to 0.1% or less in distilled water (I use 0.05%), rinse in
water and dehydrate in acetone, not alcohol, to preserve the metachromasia. You
can use various buffers to get the pH lower than 4 for even more selective
    If you are staining epoxy sections, keep the tetraborate but eliminate the
alcohol. Alcohol destroys metachromasia, at least in TB stained tissue.

Jaclynn Lett wrote:

> Regarding my Toluidine Blue stain solution, which should be staining animal
> tissue varying shades of blue-purple (which I want), but is now staining
> bright blue instead (which I don't want):
> 1% Toluidine Blue O
> 1% Sodium Tetraborate Dodecahydrate
> 30% Ethanol
> in DDI
> (1g. Tol.Blue, 1g Borax, 30mL Ethanol in 100mL DDI)
> I've received several suggestions to change the pH--some to acidic, some to
> basic.  Which direction should it be changed and what chemical do I use to
> change it?
> Thank you again,
> 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
> 314-977-0257

Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029

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