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There is little documentation of any particular specificity. It is, on the other hand, well known as a metachromatic dye and is used for general staining of semi-thin sections of epoxy and other plastic-embedded materials. The metachromatic dyes are often used to help histochemically characterize glycoaminoglycans (GAGs) and other non-neutral polysaccharides, but toluidine blue O is not usually the first choice for such purposes.
It is also widely used in the Alizarin red/Toluidine blue method for bone and cartilage. This stain also works very well with undecalcified bone as reported in J. of Histotechnology 17, 357-359 (1994).
It can be used as a surface stain, as a stain with plastic removed, or on paraffin sections.
Preparation of the stain:
Great care is needed in the mixing of the 1% toluidine blue solution. There are a number of variations on the protocol, but the following is our suggestion, at least for a starting point: