Rapidly frozen and freeze-substituted submandibular glands of young female rats were embedded in
Epon and processed for histochemical demonstration of
calcium with the
glyoxal bis (2-hydroxyanil) (
GBHA) staining method.
GBHA staining of thick
Epon sections revealed discrete
calcium reactions of moderate intensity in practically every secretory granule but not in other compartments of the acinar cells. The saliva in the excretory duct was also reactive with
GBHA and showed a drastic decrease in staining intensity toward the distal segments of excretory ducts with larger diameters. In addition, the duct saliva contained numerous tiny particles that were highly
GBHA reactive. Stromal cells and cells lining the excretory duct were totally free of reactions. In the acinar cells, X-ray analysis detected distinct peaks for
calcium in secretory granules and smaller ones in the Golgi apparatus, while they were undetectable in the rough surfaced endoplasmic reticulum (RER), implicating post-RER
calcium loading in the secretory pathway. Electron-dense deposits in the duct saliva showed distinct peaks both for
calcium and
phosphorus, though these appeared in the acinar secretory granules and other cytoplasmic regions lacked
phosphorus. Our observations thus demonstrated physiological
calcium in the intra- as well as extracellular compartments of the submandibular gland, and further confirmed drastic changes in chemical composition along the synthetic and secretory pathways of the saliva, by both histochemical and X-ray microanalytical methods.
GBHA staining of
calcium combined with X-ray microanalysis is useful for an evaluation of the physiology and histo-pathological changes of the salivary glands associated with initial phases of microliths as well as
sialoliths formation.