Phosphomolybdic and phosphotungstic acid--Victoria Blue R stains two histochemically distinct collagens: dense dark blue and loose areolar pale green.

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Abstract
Staining properties of human, dog and rat connective tissue were studied. Victoria Blue stains nuclei and elastic tissue but aqueous mounting media are required since stains are completely washed out by usual alcohol dehydration. After chroming with phosphomolybdic (PMo) or phosphotungstic (PW) acid converts the dye to an alcohol insoluble pigment. This is also insoluble in 1 N HC1 and in acid alcohol. Premordanting with PMo or PW yields a staining pattern showing the uptake localization of these acids. A 2-3 min 1% PMo or PW bath, followed by a 5 min 1% Victoria Blue R (VB) stain gives adequate coloration. Dense collagen of gastric submucosa and of derma colors dark blue; finely fibrillar areolar fibers and fat septa are light green (both stain red by Van Gieson); epithelia, keratin, gland cells and smooth and striated muscle stain light to moderate blue. Nuclei appear at most slightly deeper blue than cytoplasm, usually they are not identified. Cartilage is blue violet. Bromination, drastic acetylation, deamination and methylation inconsistently and only partially impair collagen staining. Methylation, as expected, irreversibly blocks rat mast cell staining. When the PAS reaction is performed before the PMo-VB sequence, red staining is largely suppressed; when it follows the PMo-VB reaction, it is well preserved, gastric mucin is deep red purple, basement membranes assume a deep purple tint. Cartilage may show mixed color or present blue pericellular areas in a red matrix. Interposition of the PMo-VB between the periodic acid and the Schiff reaction gives intermediate effects. Short acid dye counterstains (5-20 min) may only impair areolar tissue stains. Orange G conferred some orange to yellow on muscle and erythrocytes, eosin B and Y had less effect, and Biebrich scarlet had more. A 24 h 1% eosin gave the following: pink to red muscle, erythrocytes and epidermal keratin; light pink to pale green areolar tissue; moderate blue gastric glands and basal epidermis; and dark blue dense dermal and gastric submucosal collagen. The acid dyes probably react with the VB lake to form alcohol soluble neutral stains.