Vaginal epithelium of Herpestes elaborates neutral, sialo- and sulfomucins during anestrus. Concentration of these mucins increases during estrus and pregnancy and is highest during late pregnancy. The mucin content of the vagina is low during lactation. Intense mucification of vaginal epithelium is noticed during late pregnancy.
The cervical epithelium of Rousettus leschenaulti secretes mucins which are identified as neutral, sulfo- and sialomucins. During pregnancy the cervical epithelium undergoes mucification and an increase in mucin secretion is observed. The highest amount of mucin is present during late pregnancies of both gestations which occur in quick succession and during the lactation period of the first gestation which overlaps the estrous period of the second.
In 1970, Lojda reported the improved indigogenic method for β-D-galacto-sidase (β-gal). This method was applied to cultured skin fibroblasts from patients with three β-gal deficient disorders and from normal subjects. Cells from normal subjects showed the formation of insoluble blue indigo dye, whereas cells from the patients remained unstained. Clear-cut differences were observed in the intensity of the β-gal activities between cells from normal subjects and from the patients. This method was also applied to cultured amniotic fluid cells, and to the judgment of a genetic complementation study in restoration of β-gal activity.
We designed a modification of the aluminum-formaldehyde (ALFA) histofluorescence method based on preperfusion with buffer containing formaldehyde (FA). This modified ALFA technique enables visualization of all catecholamine (CA) neuron systems with a sensitivity comparable to that of the previously published glyoxlic acid (GA)-Vibratome method, providing better reproducibility and facility of the procedure than the ALFA method.
The peroxidase-labeled antibody method or unlabeled antibody enzyme method was used to localize tissue antigens by light microscopy on 1μm sections after fixation of 2.5% glutaraldehyde and 1% osmium tetroxide, the ultrastructure of positive-reacted cells was observed in the adjacent thin sections by electron microscopy. By means of this method, ACTH, TSH and LH were localized on 1μm thick sections of the rat anterior pituitary. At the electron microscopic level, identification of these cell types is quite different from the classical cell criteria.
Pre-embedding immunoelectron microscopy has demonstrated the ultra-structural localization of antigens in various intracellular organelles, including cisternae of rough endoplasmic reticula, pen-nuclear spaces, Golgi saccules, cytosol, secretory granules and mitochondria. In this report, major staining procedures of this method are briefly described and its applications to endocrinology are demonstrated.
Cells stained by the Grimelius or the Hellerström-Hellman silver methods or the Gomori aldehyde-fuchsin stain were compared with those stained enzyme-immunohistochemically for several peptide hormones in the same sections of normal islets and of islet cell tumors. It was found that Hellerström-Hellman positive cells were completely identical with somatostatin immunoreactive cells, and that Grimelius positive cells correlated well with glucagon, PP and gastrin immunoreactive cells, while aldehyde-fuchsin positive cells were indentical with insulin immunoreactive cells. The useful-ness of these special staining methods is discussed. Immunohistochemically, all islet cell tumors tested revealed a multicellular composition. The multicellularity of islet cell tumors and the occurrence of gastrinomas are discussed.
This is a procedure for the ultracytochemical demonstration of 3β-hydroxy-steroid dehydrogenase (3β-HSD) and glucose-6-phosphate dehydrogenase (G6PD) and the localization of these enzymes in the adrenocortical cell of rat is presented. The procedure involves pre-fixation of tissues, tissue sectioning and incubation of specimens. Brief fixation (for 30min) in a mixture of glutaraldehyde and formaldehyde (0.25%: 1% and 2%: 2%) or 2.7% glutaraldehyde was excellent to preserve both the activity of 3β-HSD and G6PD and fine cellular structure. Unfrozen sections obtained by Vibratome (Oxford) were superior to frozen sections obtained by a cryostat for preservation of the ultrastructure and enzyme activity of the cell. Sections used were 40-100μm in thickness. In the present method dehydroepiandrosterone (DHA), etio-cholane-3β-17-one (etiocholane) and pregnenolone were utilized as substrate, potassium ferricyanide as a final electron acceptor instead of tetrazolium salt, and phenazine methosulfate (PMS) as an intermediate electron carrier. As a result, the activity of 3β-HSD was localized in the cytoplasmic matrix and intracristal space of mitochondria. The activity of G6PD was visualized mainly in the cytoplasmic matrix near the plasma membrane. The reaction did not take place in any cells incubated either in the substrate-free medium or in the medium markedly inhibited by respiratory chain inhibitors such as Rotenone and Antimycin A. The findings mentioned so far were evidently those of the specific ultracytochemical reaction. Several problems will be discussed in this paper concerning the procedure for the ultracytochemical demonstration.
The ultracytochemical localization of 3β-hydroxysteroid dehydrogenase (3β-HSD) and glucose-6-phosphate dehydrogenase (G6PD) was investigated in cells of the rat adrenal cortex using copper ferrocyanide. After incubation in a 3β-HSD medium, final reaction products of copper ferrocyanide were observed in the cytoplasmic matrix near the smooth endoplasmic reticulum (SER) and in the intracristal spaces of mitochondria of the fascicular zone and reticular zone. No deposits appeared in the Golgi apparatus or nuclei. Cells of the glomerular zone were devoid of enzyme activity. The final reaction products of copper ferrocyanide of the G6PD activity were found mainly in the cytoplasmic matrix in the vicinity of the plasma membrane of the fascicular zone. Some cells of the glomerular zone demonstrated the same reaction products in the cytoplasmic matrix. This finding was consistent with the fact that G6PD is a cytoplasmic enzyme. The result of our ultracytochemical study on the localization of 3β-HSD activity has not always been in accord with the biochemical data in cell fraction reporting that most of the 3β-HSD activity is associated with the microsomal fraction.