Abstract
The staining properties of several rodent mucopolysaccharides with alcian blue and with azure A at controlled pH have been surveyed and compared with available radioautographic data of S35O4 incorporation in mucins. The effects of treatment with acid methanol and of sulfation on the staining have been determined. One sulfated, metachromatic and strongly acid type of mucin stains weakly if at all with alcian blue, remaining unstained by alcian blue in the combined alcian blue periodic acid Schiff or alcian blue-azure A (or safranin) procedures, but loses azurophilia while gaining alcian blue reactivity after mild (4 hr 37°) treatment with acid methanol. Some (probably) sulfated mucins stain fully with both dyes and others, presumably also sulfated, color only partially with azure A at pH 1.5, but maximally with alcian blue. The latter two types of substances partially or completely resist supression of either staining reaction by the "mild methylation".
A fourth category stains meta- or orthochromatically at pH 2.5 to 3, or not at all, with azure A, and colors strongly with alcian blue. Some of the latter mucins are sulfated and remain unaltered after mild treatment with acid methanol, whereas others appear to be nonsulfated and lose basophilia reversibly with such treatment. It is suggested that alcian blue staining and metachromasia or orthochromasia with azure A above pH 2.5 which is blocked after 4 hours in acid methanol at 37°C and restored by brief saponification, may be attributed to nonsulfated mucopolysaccharides containing strongly acidic carboxyls. A four hour acidic methylation at 69° eliminates nearly all the basophilia of acid mucins, and saponification restores alcian blue staining to some, but not azurophilia below pH 3.0.
Sulfation of histologic sections renders many neutral and all acid mucins fully chromotropic toward azure A at pH 1.5, but obstructs their alcian blue staining. Exposure to acid methanol after sulfation restores reactivity toward alcian blue and eliminates most artificial azurophilia; but the neutral mucins in Brunner's glands and mucous neck cells of rats and mice remain metachromatic and develop alcian blue affinity. Strong treatment with acid methanol increases the periodic acid Schiff staining of several subfated mucopolysaccharides of the rabbit. Staining with azure A or safranin after alcian blue differentiates alcian blue-negative and -positive sulfated polysaccharides.
Failure of sulfate-free acid mucins to stain with alcian blue above the pH of their dissociation and resistance of the dye complex with these mucins to dissolution by acid or alkali is interpreted as evidence against the alcian blue-substrate complex involving a salt linkage.