Histochemical staining for keratins in major salivary glands from mice and rats was compared among specimens prepared with 9 fixatives. Keratin proteins were generally confined to the striated duct (SD) and excretory duct (ED) cells in salivary glands with Carnoy's, Helly's, Bouin's, Zamboni's, formalin, alcohol and acetone fixatives and negative with PLP and Orth's fixatives. Identification of keratin proteins in both SD and GCT cells was best achieved using Helly's, Bouin's and acetone fixations. The best results of keratin reaction in SD cells, as well as histologic figures were obtained from the sections with Helly's, Bouin's, Zamboni's and acetone. For histochemical identification of keratins in GCT cells, slight positive staining was obtained in the sections using Helly's, Bouin's and acetone fixation.
Immunocytochemical detection of vimentin class of filament proteins except for keratin is reported in ductal segments of rodent submandibular glands (SMGs) and compared to stainabilities of different ductal systems; intercalated duct (ICD), granular convoluted tubule (GCT), striated duct (SD) and excretory duct (ED), and in the major salivary glands; parotid (PG), submandibular (SMG) and sublingual glands (SLG). Filament proteins; vimentin, desmin, filamin, actin and myosin were examined by immunocytochemical technique. Vimentin characteristically was confined to the GCT cells of hamster SMGs, and was distributed in luminal surface of the SD cells of guinea pigs, where it was negative in the SMGs of rats and mice, but positive in SLG duct cells. Vimentin was also positive in the ICD cells of hamster SLG. Desmin was slightly positive in the duct cells of hamsters, and intensely positive in luminal side of the SD cells of SMG and PG in guinea pigs, and of SLG in rats. Trace amounts of desmin were also noted in the GCT cells of mice and rats. Filamin was strongly positive in smooth muscles of blood vessels and trace or slightly positive in ductal cells from all the specimens. Actin was also strongly evident in smooth muscles, and slightly reactive to hamsters GCT cells, guinea pigs, and rats ductal cells and was negative in mice. Myosin was slightly positive in the GCT cells of hamsters and in the SD cells of guinea pig.
The localization of actin was investigated in various organs of rat tissues; cerebellum, cerebrum, salivary gland, pancreas, stomach, intestine, kidney, spleen, and testis by means of light microscopic histochemistry using N- (7-dimethylamino-4-methylcoumarinyl) maleimide labeled heavy meromyosin (DACM-HMM). Positive staining of actin was observed in smooth muscle cells of all organs examined, and was present in the apical part and periphery in the epithelial cells of the salivary gland, pancreas, stomach, intestine and renal tubules. Actin was also present in myoepithelial cells of the salivary gland and exocrine pancreas. It was concluded that actin is generally present not only in smooth muscle cells but also in non-muscle cells of various organs, and our histochemical method is suggested to be specific for staining actin in cells compared with immunohistologic methods.
Tissue and organ distribution of radioactive carbon from 14C-labeled galactosamine in the mouse was investigated by whole-body autoradiography and biochemical analysis. At 5 min after intraperitoneal injection of 14C-galactosamine, high radioactivity was found in the abdominal cavity, liver and urine. The radioactivities of these organs were reduced by perchloric acid treatment of sections with the exception of urine. At 30 min after injection, the radioactivity of abdominal cavity became insubstantial, but the activity in the liver, small intestine and renal cortex was maintained at a high level which still remained 1 hr after injection, and was retained considerably after the acid treatment of sections. At 6 hr, the level of radioactivity became lower in many organs except for the liver, blood and renal cortex, at which time the activity of these organs was almost completely retained even after acid treat-ent of the sections. The comparative radioactive values in organs as estimated by a liquid scintillation counter were consistent with the values estimated by densitometry of autoradiographs. In animals at 5 and 30 min after injection, paper chromatography of the acid-soluble fractions of liver and kidney had a radioactive spot for galactosamine-1-phosphate but no spot for galactosamine could be detected.
Histochemical distribution of lectin binding sites and keratin protein was studied for epithelial components, stromal elements and giant cells in calcifying epithelioma of Malherbe as well as for normal hair follicles. The HRP (horseradish peroxidase) -conjugated lectin technique was employed; the lectins used were Con A, PNA, RCA-1, DBA, SBA, WGA and UEA-1. Immunocytochemical detection of keratin protein was made using the indirect PAP technique. Basophilic cells and shadow cells in the tumor epithelial components revealed histochemical properties similar to those of matrix cells and cortex cells in growing hair follicles, respectively. Most prominent lectin binding was observed in Con A staining. In the tumor stroma, many fibrillar elements which were distinguished as immature or mature hair were found by lectin and keratin staining. Tumor giant cells showed a stronger staining for Con A and keratin, which were enhanced following treatment with amylase. The staining intensity of lectins was usually decreased in both epithelial cells and connective tissue stroma following treatment with neuraminidase.
Fine intracellular localization of acid phosphatase (AcPase) and trypsinogen was investigated in an identical specimen of rat exocrine pancreas. The tissue was embedded in acrylamide gel after AcPase enzyme histochemical reaction, and ultrathin sections were prepared successively from the gel block. The trypsinogen was detected on the cut face of each section by use of the immuno-ferritin method. AcPase activity was recognized in lysosomes, Golgi vacuoles, Golgi vesicles in trans face and in the innermost Golgi lamellae. Trypsinogen occurred in secretory granules and Golgi vacuoles. The Golgi vacuoles were classified into three types on the basis of their contents: i. e., vacuoles containing both AcPase activity and trypsinogen-immunoreactivity, vacuoles containing only tryp-sinogen-immunoreactivity, and vacuoles showing only AcPase activity. The first type of the Golgi vacuoles was most frequently observed and the third type of Golgi vacuoles, rarely. On the basis of the result, it is concluded that lysosomes and secretory granules develop simultaneously from the trans face of the Golgi apparatus. It is suggested that one role of lysosomal enzymes may be intracellular modification of secretory proteins in the Golgi mature face of rat exocrine pancreas.
The localization of calmodulin in guinea pig testis and spermatozoa was determined by both immunoperoxidase labeling and immunocolloidal-gold methods on the electron microscopic level. In spermatids, reaction deposits were scattered all over the cytoplasm, but hardly recognized in acrosome vesicle and Golgi complex. In the neck portion of elongated spermatids, anti-calmodulin immunoreactive sites were diffusely localized in cytoplasm, and also recognized along the axial filaments and near the connecting pieces. Mature spermatozoa exhibited the following distinct regions of anti-calmodulin immunoreactive sites; the cytoplasm between plasma and outer acrosomal membranes, between nuclear envelope and inner acrosome membrane, along the axial filaments, and outside of fibrous sheath. These findings suggest that calmodulin may play a role in both acrosome reaction and tail movement.
The effects of testosterone and 17β-estradiol on uterine thymidine kinase (TK) activity in immature rats were investigated. Injection of either compound alone resulted in a more than 30-fold increase in TK activity 30 hr later. Injection of the two compounds together resulted in an even greater increase in TK activity and in the activities of its isozymes separated by DEAE cellulose column chromatography. Autoradiographic examination showed that testo-sterone alone induced marked DNA synthesis in the endometrial stroma and myometrium, but not in the endometrial epithelium, and that its effect on DNA synthesis in all these tissues was enhanced by the additional injection of 17β-estradiol.
Purified schizophyllan (SPG), which was not labeled with radioactive isotopes, was administered intraperitoneally or intramuscularly to female mice of the ICR and DBA strains every third day from the 13th day after sarcoma-180 inoculation to the 28th day. Four days after the last SPG administration, 400 μCi/head of 3H-SPG was injected intravenously. The liver, spleen, bone marrow, lymph nodes, thymus, and tumor were studied with whole-body, microscope, and electron microscope (EM) autoradiography (ARG). Anesthetized animals were used for whole-body ARG after tissue pieces were removed and fixed for micro-ARG and EM-ARG. In whole-body ARG, 3H-SPG was mainly accumulated in the liver, spleen, lymph nodes, thymus, bone marrow, and a capsular belt area of the tumor. In the tumor capsular belt area, a very strong accumulation was observed in ICR mice, but the accumulation was very weak in DBA mice. In micro-ARG, 3H-SPG was incorporated in the reticuloendothelial cells; Kupffer's cells in the liver, and reticulum cells and macrophages in the spleen, bone marrow, lymph nodes, and thymus. A large number of macrophages in the tumor capsular belt area were observed migrating between the tumor cells and 3H-SPG was strongly incorporated in their cytoplasm in ICR mice. But only a few macrophages were observed there in DBA mice. In EM-ARG, 3H-SPG was accumulated in special lysosomal granules in the reticuloendothelial cells, including Kupffer's cells and macrophages. The special granules were enveloped with a limiting membrane, and were composed of a compact fine filamentous substance and an electron opaque lysosomal cap structure (filamentous ball, Fb). The nature of these fine Fbs was exactly the same as that of SPG macromolecules observed in the negatively stained samples. There were no great differences on the observations by ARG and histology between the SPG-treated ICR and DBA mice, except in the tumor region. A large number of macrophages with many intracytoplasmic Fbs were migrating into the sarcoma nest in the capsular area, and many sarcoma cells were rapidly degenerating or had disappeared in the SPG-treated ICR mice. In DBA mice, however, there were no significant changes in the cancer nests; the cancer cells were growing and increasing rapidly, and the animals died within a month. These autoradiographic data suggest that the SPG granules (Fbs) containing activated macrophages in the ICR tumor nests play an important role in destroying the growing sarcoma cells, but there was no evidence of active macrophages in the DBA sarcoma nests. The Fbs in the macrophages in ICR mice probably produce a large amount of various lysosomal enzymes and act as activators of the destruction of sarcoma cells, but did not act in DBA mice. The reason for such a big difference between the two mouse strains was not clear, but some immunogenomic factors could be concerned.