The high iron diamine (HID) method has been found to impart density at the ultrastructural level selectively to sites known to contain sulphated complex carbohydrates. Thus, immature primary granules in rabbit heterophils, immature precrystalloid granules in rabbit eosinophils, all granules of rabbit basophils, mouse and rat mast cells and the nucleoids of alpha-granules of rabbit platelets were stained by HID. Granules of mast cells in rat cervical lymph node varied in the distribution pattern of the HID-reactive component. Mucous droplets within goblets of mouse colonic epithelial cells varied in HID reactivity. Sites known to contain sialomucin but no sulphates, such as mucous cells and apical plasmalemmae in mouse rectosigmoid colon, failed to stain with HID in contrast to their reactivity of dialysed iron at the ultrastructural level. The surface of mast cells and blood cells lacked affinity for HID, indicating that the dialysed iron binding at the surfaces can be attributed to neuraminic acid. HID proved more effective than dialysed iron in visualizing acid mucosubstance in precursor forms of the crystalloid granules in the eosinophil and in mast cell granules. Inclusion of 0.5% glycerol in the HID solution enhanced staining in mouse colon.

Application of cytochemical methods for complex carbohydrates at the light and electron microscopic levels served further to differentiate secretory cells of the rat tracheobronchial surface epitheliuim into serous and mucous categories, and permitted subclassification of serous cells into four types and of mucous cells into three types. The granules in different serous cells and Clara cells varied in staining with the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method, but generally lacked dialyzed iron (DI) affinity and apparently contained neutral glycoprotein. However, in some focal areas, serous and Clara cell granules showed a pattern of DI staining similar to the PA-TCH-SP reactivity. The PA-TCH-SP and DI methods stained granule glycoconjugate comparably in the three mucous cell types. The lack of high iron diamine (HID) affinity in these sites testified to carboxylated, periodate-reactive mucosubstance, presumably sialylated glycoprotein. The luminal surface of the apical plasmalemma of all surface epithelial cells stained with DI but lacked HID affinity. The PA-TCH-SP technique stained the surface of the apical plasmalemma of mucous cells, serous cells, and Clara cells in decreasing order of intensity. Microvilli of cililated cells were PA-TCH-SP-positive, but cilia were unreactive and apparently contained a glycosaminoglycan rather than the presumed sialylated glycoprotein of the other surfaces. DI and PA-TCH-SP reactivity of the apical far exceeded that of the basolateral plasmalemma in all surface epithelial cells. Two strata, presumed to be the lamina lucida and the lamina diffusa of the basement membrane under all the surface epithelial cells, stained with the DI but not the PA-TCH-SP method and apparently contained glycosaminogycan. Intraepithelial cells, interpreted as globule leukocytes, appeared to represent mast cells infiltrating the epithelium and carrying out endocytic activity.

Cytochemical methods applied to examination of rat respiratory tract glands have revealed diversity of secretory complex carbohydrates. With the Alcian blueperiodic acid Schiff (AB-PAS) and the high iron diamine (HID) techniques at the light microscopic level, certain patterns of glycoprotein content were noted at various levels of the respiratory tract. Serous tubules and demilunes found in abundance in laryngeal and tracheal glands contained neutral glycoprotein. Mucous tubules found in abundance in epiglottal and laryngeal glands and in lesser number in tracheal glands most often produced sulfated glycoprotein. However, mucous tubules in epiglottal and tracheal glands contained a few cells with sialylated glycoprotein, and mucous tubules in some areas in laryngeal glands contained mainly cells producing sialylated glycoprotein. Mucous ducts found in abundance in lower laryngeal and tracheal glands formed mainly sialylated glycoprotein and contained infrequent cells with sulfated glycoprotein. The type of glycoprotein found in each cell type by light microscopy was confirmed at the ultrastructural level by the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP), dialyzed iron (DI) and high iron diamine (HID) methods. Serous cell granules displayed light reactivity with the PA-TCH-SP method and no DI or HID affinity and were judged to contain sparse neutral glycoprotein. Serous granules disclosed negative central foci with the PA-TCH-SP method. Granules of most mucous tubule cells stained strongly with the PA-TCH-SP and HID procedures and contained abundant sulfated glycoprotein. Occasional mucous tubules stained with the PA-TCH-SP but not with the HID method and apparently corresponded with cells judged to form sialylated glycoprotein from their blue staining with the HID-AB sequence. Two zones within individual granules in some cells revealed different HID staining intensity and appeared to differ in the amount or kind of sulfated glycoprotein they contained. Some cells exhibited granule heterogeneity containing both HID-positive and unstained granules. Spherical cores present in granules of mucous tubules below the upper laryngeal level occasionally appeared bizonal and invariably lacked reactivity demonstrative of complex carbohydrate. Mucous duct cell granules stained heavily with the PA-TCH-SP and DI methods and reacted infrequently with the HID procedure and were considered generally to contain sialylated glycoprotein, and occasionally to form sulfated glycoprotein. The three carbohydrate stains distinguished a heavily and a moderately reactive zone in the cortex outside the monophasic or biphasic, carbohydrate-free cores in granules of some mucous duct cells.Respiratory diseases are often associated with hypersecretion of mucus, but little is known of the composition of the stored mucus secretion in normal or disease states. Examination of the rat respiratory surface epithelium by ultrastructural methods for differentiating complex carbohydrates has shown a diversity of secretory cells...

The structure of rat respiratory glands has been investigated by light and electron microscopy. The glands exhibited a tubulo-acinar organization. Individual secretory units were composed of serous tubules or serous demilunes and mucous tubules with narrow lumens at the blind end (proximally), and of mucous ducts with wide lumens which opened to the tracheal lumen distally. Near the junction of the duct with the trachea, mitochondria-rich cells and a few ciliated cells were interspersed with mucous cells. It was found that the histology as well as the ultrastructure of the secretory product of these gland cells varied depending upon the location of the glands in the airway. For this reason, the glands of the epiglottis, larynx and trachea have been described separately. Epiglottal glands consisted of many mucous tubules, a few serous elements and an occasional mucous duct. Laryngeal glands contalned many serous and mucous tubules and a few mucous ducts. Tracheal glands were composed of serous tubules, a few mucous tubules and prominent bucous ducts. Serous tubule cell granules formed a uniform population within a cell profile but varied in cells of the epiglottis, compared with those of the larynx and trachea. Granules in mucous tubule cells differed from those of serous cells. Mucous cell granules also appeared uniform within a single cell but differed from cell to cell and at different levels of the respiratory tract. A particular granule type was predominant at each level. Cells of mucous ducts differed somewhat from those of mucous tubules and comprised two general secretory cell populations. Proximal mucous duct cells contained abundant granules that generally resembled those of mucous tubule cells but were localized in two areas of the cytoplasm. Dilated cisternae of rough endoplasmic reticulum with a bizonal content similar in density of zones seen in mature granules further characterized proximal mucous duct cells. The distal mucous duct cell generally contained sparse apical granules and lacked dilated rough endoplasmic reticulum.

The present study has applied the low iron diamine (LID) method at the ultrastructural level to demonstrate acid glycoconjugates. We have examined rat epiphyseal cartilage, human bone marrow, rat tracheal glands, and mouse sublingual glands stained with LID prior to embedment. The LID staining appeared to require postosmication for adequate visualization at the electron microscope level. Thiocarbohydrazide-silver proteinate (TCH-SP) staining of thin sections variably enhanced LID reactive sites. LID-TCH-SP stained carboxyl and sulfate groups of glycosaminoglycans in the extracellular cartilage matrix, secretory granules, and expanded Golgi saccules of chondrocytes. In human bone marrow, LID-TCH-SP variably stained the cytoplasmic granules, known to contain sulfated glycosaminoglycans, and the external surface of the plasma membrane of leukocytes. Moderately strong LID staining was observed in secretory granules in mucous tubules of rat tracheal glands, known to contain sulfated glycoproteins, and in acinar cells of mouse sublingual glands, known to contain a sialoglycoprotein. The lack of sulfated glycoconjugates in acinar cells of the mouse sublingual gland was confirmed by their failure to stain with the high iron diamine method. Thus these studies indicate that the LID and LID-TCH-SP methods are useful for the ultrastructural localization of carboxylated and sulfated glycoconjugates in extracellular and intracellular sites.