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Distribution of glycoconjugates in different areas of the rat kidney was studied by light and electron microscopy using six different horseradish peroxidase-labeled lectins. Glomeruli and brush borders of the proximal tubules reacted differently to these lectins, which indicated differences in the carbohydrate compositions of those regions. The ascending limb of Henle's loop (ALH) had strong binding sites for peanut agglutinin (PNA) and soybean agglutinin (SBA). Dolichos biflorus agglutinin (DBA) did not stain the cells of ALH but did stain those of distal convoluted tubules (DCT). DBA is a good marker for distinguishing ALH from DCT. DBA, PNA, and SBA were also good markers of the collecting duct. Ricinus communis agglutinin (RCA-1) and wheat germ agglutinin (WGA) diffusely stained the various components of different parts of the kidney.

We evaluated homeostatic mass control in non-neoplastic gastric epithelia under Helicobacter pylori (HP) infection in the macroscopically normal-appearing mucosa resected from the stomach with gastric cancer, immunohistochemically analyzing the proliferation, kinetics of stem cells and programmed cell death occurring in them. Ki67 antigen-positive proliferating cells were found dominantly in the elongated neck portion, sparsely in the fundic areas and sporadically in the stroma with chronic infiltrates. CD117 could monitor the kinetics of gastric stem cells and showed its expression in two stages of gastric epithelial differentiation, namely, in transient cells from the gastric epithelial stem cells to the foveolar and glandular cells in the neck portion and in what are apparently progenitor cells from the gastric stem cells in the stroma among the infiltrates. Most of the nuclei were positive for ssDNA in the almost normal mucosa, suggesting DNA damage. Cleaved caspase-3-positive foveolar cells were noted under the surface, suggesting the suppression of apoptosis in the surface foveolar cells. Besides such apoptosis of the foveolar cells, in the severely inflamed mucosa apoptotic cells were found in the neck portion where most of the cells were Ki67 antigen-positive proliferating cells. Beclin-1 was recognized in the cytoplasm and in a few nuclei of the fundic glandular cells, suggesting their autophagic cell death and mutated beclin-1 in the nuclei. Taken together, the direct and indirect effects of HP infection on the gastric epithelial proliferation, differentiation and programmed cell death suggested the in-situ occurrence of gastric cancer under HP infection.

Summary Background Fabry disease is characterized by the systemic accumulation of glycosphingolipids, particularly in the lysosomes of vascular endothelial cells of most organs due to the deficient activity of α-galactosidase A. The major glycolipid accumulated in tissue is globotriaosylceramide (GL-3). To date, no direct detection of GL-3 by immunoelectron microscopy has been reported. Objectives To examine whether GL-3 is accumulated exclusively in lysosomes of cutaneous cells using an anti-GL-3 monoclonal antibody (mAb) and immunoelectron microscopy. Methods Skin specimens from seven patients with Fabry disease were examined immunohistochemically by light and electron microscopy using an anti-GL-3 mAb. Results By light microscopy, the cytoplasm of vascular endothelial cells, eccrine gland cells, and perineurium was stained with mouse anti-GL-3 antibody. Electron microscopically, positive signals for GL-3 were limited to dilated lysosomes in the cytoplasm of endothelial cells, pericytes, eccrine gland cells, dermal fibroblasts and perineurium. Conclusions Our results demonstrate that the cytoplasmic deposit in Fabry disease was GL-3 and the accumulated GL-3 was localized essentially to lysosomes.

Gastric parietal cells are characterized by intracellular canaliculi with many microvilli and tubulovesicles. These membranous structures are continuous and change in volume according to gastric acid secreting or resting state. Morphological changes are induced by cytoskeletal actin. Actin filaments are anchored to the cell membrane via ezrin. This study aimed to ascertain whether morphological changes were related to the nature of ezrin molecules, which were either phosphorylated or non-phosphorylated. Rat stomach tissues were rapid-frozen followed by freeze-substitution and embedding in paraffin for light microscopy. Also, the specimens were high pressure-frozen, freeze-substituted and embedded in Epon 812 or Lowicryl K4M for electron microscopy. Sections from paraffin or Lowicryl K4M were stained with anti-H+·K+-ATPase antibodies, anti-phosphorylated or non-phosphorylated ezrin antibodies, and anti-actin antibody. The H+·K+-ATPase was distributed on the intracellular canaliculi and tubulovesicles. The intracellular canaliculi were labeled with anti-phosphorylated ezrin antibodies in fed (gastric acid secreting state) rats. These were stained weakly in starved (gastric acid resting state) rats. Anti-nonphosphorylated ezrin antibodies hardly stained these organelles in either state. A possible explanation is that the phosphorylation of ezrin molecules is related to the reciprocal membranous transformation between intracellular canaliculi and tubulovesicles.

To understand proliferation and suppression in ameloblastoma that exhibit locally invasive growth and recur clinically, the expression of proliferating cell nuclear antigen (PCNA), Ki-67 antigen, topoisomerase IIα, p53 and p21 proteins were analyzed with immunohistochemistry. Sections of archival ameloblastoma tissues were used (16 plexiform and 14 follicular types). Each antigen was labeled by the polymer method with optimal antigen retrieval. The plexiform type exhibited higher expression of PCNA, Ki-67 and topoisomerase IIα antigens than the follicular type. Expression of PCNA correlated significantly with that of the Ki-67 antigen. The plexiform type also exhibited greater expression of p53 and p21 proteins than the follicular type. Expression of p53 protein correlated significantly with that of p21, PCNA, Ki-67 antigen and topoisomerase IIα. Expression of p21 protein did not correlate with that of the proliferation-related antigens. These findings suggest that the plexiform ameloblastoma has higher proliferating activity and malignant potentiality as neoplastic cells than the follicular ameloblastoma.

Gastric parietal cells were examined for changes in their ultrastructure and distribution of the proton pump during feeding and fasting states in rats. The fundic glands from rats fed ad libitum or fasted with free access to water were cryofixed using high-pressure freezing followed by freeze-substitution in acetone containing osmium or acrolein and then embedded in Epon 812 or Lowicryl K4M resin, respectively. Excellent ultrastructural preservation was achieved. During the feeding state, intracellular canaliculi and numerous microvilli were well developed, while tubulovesicles were poorly developed. In contrast, during the fasting state, the microvilli in the narrowed space of the intracellular canaliculi were tightly packed and the tubulovesicles were enlarged. Ultrathin sections were immunostained with antibodies against the alpha- and beta-subunits of the proton pump, H+ x K(+)-ATPase, using the immunogold method. The labelling was strong and clearly localized in comparison with that obtained using the conventional chemical-fixation method. Each subunit was localized on the membrane of the microvilli, intracellular canaliculi and tubulovesicles. The distribution of subunit proteins varied between the two states. During ad libitum feeding, the immunolabelling was localized strongly on the membranes of the microvilli and intracellular canaliculi. In contrast, the labelling was strong on the tubulovesicle membrane in the fasting state. The results obtained with each anti-subunit antibody by H+ x K(+)-ATPase immunostaining revealed differences in distribution and labelling density between the feeding and fasting states.

Anti-Helicobacter pylori (HP) therapy succeeded in regressing gastric B-cell lymphomas (gBL) with a close relation to infestation of HP, although the exact mechanism of anti-HP therapy in gBL has not yet been clarified. In order to see a part of the mechanism, this study analyzed stromal cells in 34 gBL cases comprised of 11 cases of mucosa-associated lymphoid tissue (MALT) type and 24 cases of diffuse large B-cell lymphoma (DLBL) by means of immunohistochemistry of CD3, CD5, CD79a, CD68, CD21, S100 protein, thymidine phosphorylase (TP) and inducible nitric oxide synthase (iNOS). Numerous CD68-positive stromal cells were seen in 9 cases of MALT type and 22 cases of DLBL. Many dendritic cells (DC) expressed TP and formed an ill-defined meshwork in the background in 8 cases of MALT type and 17 cases of DLBL. In most cases, transition of CD68-positive stromal cells to DC expressing TP was recognized. There was more or less intermingling of CD3-positive T-cells in all cases. Expression of iNOS was seen in some stromal cells and glandular epithelial cells, but only in 3 cases of MALT type. In the germinal center (GC) colonization of MALT type there were CD21-positive follicular DC, CD68-positive stromal cells, TP-expressing DC and iNOS-expressing cells; whereas in that of DLBL the meshwork of CD21-positive follicular DC was destroyed and the stromal cells did not express TP or iNOS. Then, a peculiar co-existence of intermingling T-cells, CD68-positive stromal cells, DC expressing TP forming an ill-defined meshwork, and lymphoma cells were recognized in gBL. A small number of stromal cells and glandular epithelial cells expressed iNOS and prepared a nitric oxide-rich microenvironment for growth and transformation of MALT type lymphoma cells. This peculiar histogenesis of gBL was thought to explain a part of the mechanism of the anti-HP therapy against gBL.

Gingival fibromatosis is a rare disease characterized by enlargement of the gingiva. The purpose of this study was to analyze a case of idiopathic gingival fibromatosis, using histochemical and immunohistochemical staining and transmission electron microscopy. The patient was a 39-year-old Japanese man, in whom the gingiva was enlarged throughout the entire mandible and maxilla. Specimens of gingival fibromatosis exhibited epithelial hyperplasia and increased amounts of collagen fiber bundles in the connective tissue light-microscopically. Well-developed collagen bundles were strongly stained with Azan and Masson trichrome staining. Immunohistochemically, the gingival connective tissue was specifically stained by type I collagen and vimentin antibodies. Ultrastructurally, the lesion consisted of fibroblasts and mature collagen fibers running in all directions. No myofibroblasts were detected histochemically, immunohistochemically, or ultrastructurally. These findings suggested that this disease may be the result of an increase in collagen synthesis by the fibroblasts and/or that it may be associated with one of the findings of histologic heterogeneity.

Alpha-N-acetylgalactosaminidase (alpha-NAGA) deficiency with angiokeratoma corporis diffusum (AKCD) is one of the lysosomal storage diseases. GalNAc(alpha))1-O-Ser/Thr (Tn) is theoretically deposited in lysosomes, but substances with attached galactose and neuraminic (sialic) acid (T and sialosyl Tn, respectively) are excreted in patients' urine. In this study, in two Japanese cases we analyzed the material accumulated in lysosomes using immunoelectron microscopy with mouse antibodies to Tn, sialosyl Tn and T (Thomsen-Friedenreich) antigens in order to find out what substance(s) is really deposited in lysosomes. We found that only GalNAc(alpha)1-O-Ser/Thr (Tn) was actually accumulated in vacuolated lysosomes of vascular endothelial cells, eccrine sweat gland cells, fibroblasts and pericytes. Galactosylation and sialylation of Tn appears to occur in cells other than those in the skin. The results suggest that this disease is caused by a single enzyme deficiency.

Gastric gland component cells were electron-microscopically and immunoelectronmicroscopically examined with high-pressure freezing followed by freeze substitution and a low-temperature embedding resin method and compared to that of the conventional chemical-fixation method. The rat gastric gland was high-pressure frozen, freeze-substituted with acetone-containing osmium or acrolein, and embedded in Epon 812 or Lowicryl K4M, respectively. Using the high-pressure freezing method, the vitreous freezing range reached the depth of 150 microns from the surface. The ultrathin sections from both procedures embedding in Epon 812 and Lowicryl K4M were doubly stained with uranyl acetate and lead acetate, and histochemically or immunohistochemically stained, respectively. In comparison to the conventional chemical fixation method, excellent results were obtained with respect to ultrastructural preservation. The stainings performed in this experiment included periodic acid-thiocarbohydrazide-silver proteinate staining, cationic colloidal cold at pH 2.5 staining, Helix pomatia lectin-staining, anti-alpha or -beta subunit antibodies of H+K(+)-ATPase immunostaining and pepsinogen immunostaining. The staining intensity of those was stronger than that of the conventional immersion-chemical fixation method. In addition to these results, the labels also showed good specific localization. In this paper, we provide a description of the high-pressure freezing followed by freeze substitution and low-temperature embedding resin method compared to the conventional chemical-fixation method. Our results suggest that this method is a suitable tool for ultrastructural and histochemical/immunohistochemical studies at high resolution.

Inducible nitric oxide synthase (iNOS) is expressed in the germinal centers (GCs) of mucosa-associated lymphoid tissue (MALT) and regional lymph nodes (RLNs) in the stomachs of patients with Helicobacter pylori (HP)-related ulcer. This study used immunohistochemistry to deduce how the iNOS was expressed and what the iNOS induced on the MALT in four cases. HP bodies were labeled with anti-HP, anti-Lewis X and Y, and anti-IgH antibodies in the surface mucous coat and in the glands. Lewis X or Y was detected in regenerative, metaplastic and atrophic glandular epithelial cells and GCs of RLNs. The expression of iNOS was recognized again in some glandular epithelial cells and in the GCs of the MALT and RLNs. The developed MALT included many IgM + CD5 - cells in the background of an ordinary mucosal immunity. Pseudonodular growth of the IgM + CD5 - cells was recognized in one case. It is suggested that Lewis X and Y from HP bodies induced iNOS in the GCs. Dominant IgM + CD5 - cells in the MALT suggested that excess nitric oxide (NO) produced by iNOS disturbed anti-HP B-cell immunity development in the GCs. As NO is also a mutagen, the IgM + CD5 - B-cells' pseudonodular growth might explain MALT-type lymphomagenesis.

Epithelial growth factor receptor (EGFR) sends signals to the proliferation signal transduction system, receiving two ligands: epithelial growth factor (EGF) and transforming growth factor-alpha (TGF-alpha). This immunohistochemical study examined the roles of EGFR and its ligands in the proliferation of normal and neoplastic vulvar squamous cells in 25 patients with vulvar squamous cell carcinoma (VSCC), 10 patients with vulvar condyloma acuminata (VCA), 15 patients with vulvar intra-epithelial neoplasm I-II or III (VIN I-II or III), and 5 subjects with vulvar normal squamous cells (VNSC). EGFR was detected in a few basal cells in 40% of the VNSC, in highly dysplastic cells in 40% of the VIN III, in many neoplastic cells in 80% of the VCA, and in some malignant cells in 64% of the VSCC. EGF was seen in the cytoplasm in 20% of the VIN I-II, 100% of the VIN III, 100% of the VCA, and 100% of the VSCC. Diffuse TGF-alpha was weakly expressed in the cytoplasm in 100% of the VNSC, more intensely in 100% of the VIN and 100% of the VCA, and intensely in 100% of the VSCC. These findings led to the suggestion that the TGF-alpha-EGFR system maintains the growth of normal squamous cells and, in part, maintains the growth of dysplastic and neoplastic squamous cells in the vulva. EGF expression was an early sign of neoplasia. The expression of EGFR with overexpression of its two ligands contributed to the proliferation of dysplastic and neoplastic squamous cells in VIN III and VCA. EGFR expression appeared to contribute to essential neoplastic abnormalities in 64% of the VSCC.

The proton pump H+-K+-ATPase is the final common pathway mediating the production and secretion of hydrochloric acid by gastric parietal cells. The present studies were undertaken to examine whether the expression of gastric H+-K+-ATPase mRNA and protein changes are associated with the development of H+-K+-ATPase activity in the rat fundic gland. H+-K+-ATPase activity was examined in rat fundic gland at different stages from gestational day 18.5 to postnatal 8 weeks. The expression of H+-K+-ATPase mRNA was detected by in situ hybridization using a digoxigenin-labelled RNA probe with a tyramide signal amplification system. The expression of H+-K+-ATPase protein was evaluated by immunoblotting and immunohistochemistry using antibodies against H+-K+-ATPase alpha- and beta-subunits. We found that H+-K+-ATPase enzyme activity was detectable from the onset of gland formation (day 19.5 of gestation) and increased with age in the developing rat fundic gland. Expression of mRNA and protein was also discernible at the same time, and a progressive increase in expressions was observed as rats developed. Our results suggested that in developing rat fundic gland, the expression of both mRNA and protein of H+-K+-ATPase increased with age in a manner that parallels the development of H+-K+-ATPase enzyme activity.

BACKGROUND MUC1 mucin is a transmembrane, mucin-like glycoprotein encoded by the MUC1 gene. Although MUC1 expression has been identified in a variety of neoplastic tissues, to the authors' knowledge, few studies have examined MUC1 expression in premalignant and malignant oral lesions. METHODS A total of 36 specimens of oral epithelial dysplasia, 12 carcinoma in situ (CIS) specimens and 77 specimens of oral squamous cell carcinoma (OSCC), were examined by both light and electron microscopy using immunohistochemical staining of MUC1 mucin. Relations between staining patterns and clinicopathologic findings also were examined. RESULTS Distinct membrane MUC1 mucin staining patterns were identified in epithelial dysplasia (33.0%), CIS (50.0%), and OSCC (59.7%) cases. A predominantly cytoplasmic staining pattern was detected in epithelial dysplasia (5.6%), CIS (41.7%), and OSCC (32.5%) cases. Significant positive correlations were found between MUC1 mucin membranous immunoreactivity and disease progression from epithelial dysplasia to OSCC (P < 0.01), mode of tumor invasion (P < 0.02), and lymph node metastasis (P < 0.01). Furthermore, the malignant transformation of oral epithelium, tumor invasion, and tumor metastasis were associated with higher MUC1 mucin expression in the cytoplasm (P < 0.01). In addition to the usual cell surface expression, cytoplasmic expression of MUC1 mucin was confirmed by colloidal gold labeling with transmission electron microscopy. CONCLUSIONS The results of the current study suggest that determination of MUC1 mucin expression may be a parameter in the diagnosis of premalignant and malignant lesions arising in the oral cavity and that this expression may affect the malignant behavior of OSCC. MUC1 mucin expression may be a useful diagnostic marker for prediction of the invasive/metastatic potential of OSCC. Cancer 2000;88:245–54. © 2000 American Cancer Society.

The development of rat fundic gland was studied by immunohistochemistry using a recently developed monoclonal antibody, HIK 1083, at both light and electron microscope levels. Antibody HIK 1083 recognized oligosaccharides with a non-reducing terminal alpha-linked N-acetylglucosamine (GlcNAc) residue. In the developing rat fundic gland, cells expressing alpha-GlcNAc residues were discernible from day 19.5 of gestation and continued to exist till adult. The distribution of the alpha-GlcNAc expressing cells was consistent with that described previously for cells reacting to Griffonia simplicifolia lectin (GSA-II) in all developmental stages. These cells were located at the bottom of the fundic gland when they first appeared. With the elongation and maturation of the gland, these cells moved upwards and were finally restricted in the neck region of the gland. Combining previous reports and the present electron microscopical observations, HIK 1083-positive cells in the adult rat fundic gland are mucous neck cells. The interaction between antibody HIK 1083 and GSA-II lectin was investigated. GSA-II prevented the subsequent binding of HIK 1083, while HIK 1083 did not prevent GSA-II binding to mucous neck cells. Our results suggested that alpha-GlcNAc residues exist in rat fundic gland from day 19.5 of gestation and continue to exist till adult. Cells expressing alpha-GlcNAc residues appeared as typical mucous neck cells from postnatal four weeks.

We investigated mucin 1(MUC1) expression in 10 vulvar condyloma acuminata(VCA), 15 vulvar intraepithelial neoplasia(VIN) and 30 vulvar squamous cell carcinomas (VSCC) using three different monoclonal antibodies(Ma695, CA15−3, and DF3), and compared the level of MUC1 expression with the clinicopathologic features of VSCC. Of the three monoclonal antibodies, Ma695 showed the strongest immunoreactivity. MUC1 expression levels were high in VSCC, low in VIN III, and undetectable in VIN I-II, VCA and normal vulvar epithelium. Patients with human papilloma virus(HPV)−negative VSCC showed higher MUC1 expression than HPV−positive patients. The MUC1 expression level gradually increased from well through moderately to poorly differentiated VSCC, and there was a statistically significant difference between moderately/poorly differentiated VSCC and well differentiated VSCC(p

In the gastrointestinal tract, Class III mucinsecreting cells include the cardiac mucous cells, mucous neck cells, pyloric gland cells and Brunner's gland cells. It has been found that these cells are stained by a recently developed monoclonal antibody, HIK 1083, at the light microscopic level. In the present study, we compared HIK 1083 staining with paradoxical Concanavalin A type III staining (PCS) and GSA-II lectin staining, and found that all three methods revealed the same reactivity at both the light and electron microscopic levels. HIK 1083 staining exhibited highly selective reactivity with cardiac mucous cells, mucous neck cells, pyloric gland cells and Brunner's gland cells. These results indicate that mucus-secreting cells of foregut origin share a common lineage and contain the same cell phenotype. Class III mucin secreted by these cells was characterized by the presence of a peripheral α-GlcNAc residue on the carbohydrate moiety of its constituent glycoproteins. Because of the ease and specificity of immunostaining using monoclonal antibody HIK 1083, it could replace PCS staining for both light and electron microscopy.

Activins are multifunctional proteins that belong to the transforming growth factor-beta superfamily and are thought to play an important role in modulating the formation of bone. Activins exert their cellular effects by way of activin type-I and type-II serine/threonine kinase receptors. Follistatin is an activin-binding protein that can suppress the biological effects of activins. In this study, the immunohistochemical expression of activin A, follistatin, and activin receptors was studied during fracture healing in the rat. Activin A was weakly detected in the periosteum near the fracture ends at an early stage but was absent in the chondrocytes around the fracture gap, where endochondral ossification took place. An antibody to follistatin stained osteogenic cells in the periosteum near the fracture ends; moderate and strong staining were observed in proliferating, mature, and hypertrophied chondrocytes at the sites of endochondral ossification. Levels of activin A and follistatin were high near the osteoblasts on the surface of the newly formed trabecular bone. In addition, an intense localization of activin A was noted where multinucleated osteoclast-like cells were present. This study suggests that the activin-follistatin system may contribute to cellular events related to the formation and remodeling of bone during fracture healing. Activin type-I and type-II receptors were co-expressed in intramembranous and endochondral ossification sites. The expression of activin type-I, type-II, and type-IIB receptors in the absence of activin A in the endochondral ossification suggests that other isoforms of activins may signal by way of these receptors.

Bone marrow embedding in the hydrophilic resin, Lowicryl K4M, followed by cationic colloidal gold (CCG, pH 1.0) staining was used to study the sulfated glycosaminoglycans (GAGs) and their sites of sulfation ultrastructurally in various maturational stages of both basophil granulocytes and basophil granules in the guinea pig. CCG at pH 1.0 is specific for sulfated GAG staining. Basophil granulocytes and granules reacted positively to CCG with a variety of staining according to the stage of maturation. The formation of basophil granules takes place throughout the myelocyte stage. Early basophil myelocytes contain a large Golgi apparatus with active granulogenesis, while late myelocytes contain a small and less active Golgi apparatus as judged by CCG staining. All the immature granules and some of the granules with characteristic ultrastructure stained positively. However, some of the mature granules had lost their affinity for CCG upon maturation. Interestingly, strongly positive CCG staining was also observed in the trans to transmost Golgi apparatus. This indicates that sulfation of GAGs occurs in the trans to transmost Golgi apparatus in all maturational stages of basophil granulocytes. Treatment with chondroitinase ABC or heparinase I abolished the majority of CCG staining.

Using bone marrow embedded in hydrophilic resin Lowicryl K4M and cationic colloidal gold pH 1.0 labelling, we studied sites of sulphation and sulphated glycosaminoglycans ultrastructurally in various maturational stages of both eosinophil granulocytes and eosinophil granules of guinea pig. Eosinophil granules reacted positively to cationic gold, the pattern of labelling varying according to the degree of cell maturation. The formation of eosinophil granules takes place throughout the myelocyte stage. Early eosinophil myelocytes contain a large Golgi apparatus with active granulogenesis, while late ones contain a small and less active Golgi apparatus. All the immature granules were labelled positively. However, mature granules with a central crystal bar lost their affinity towards colloidal gold. Interestingly, strong colloidal gold labelling was also observed in the trans to transmost Golgi apparatus, especially in immature eosinophil granulocytes. This indicates that sulphation of glycosaminoglycans occurs in the trans to transmost Golgi apparatus of eosinophil granulocytes. Prior absorption with poly-L-lysine prevented colloidal gold labelling of tissue sections. Methylation of sections at 37°C did not alter the gold labelling, whereas the labelling disappeared after methylation at 60°C. Prior treatment with chondroitinase ABC or heparinase I abolished the majority of colloidal gold labelling in immature eosinophil granules. Taking these results together, we conclude that immature eosinophil granules contain sulphated glycosaminoglycans including chondroitin sulphate or heparan sulphate or both.

We developed a novel technique to detect S-phase skin cells by applying bromodeoxyuridine (BrdU) epicutaneously using an occlusive dressing (OD) method. BrdU was scarcely absorbed from the skin with a simple epicutaneous application, whereas the incorporation of BrdU was very well promoted with the use of our OD method. We applied BrdU on the backs of rats using this method and investigated the conditions required for an optimal response, with a special focus on the period of application, the concentration of BrdU used and vehicles suitable for the immunocytochemical staining of this agent. From these experiments, we were able to detemine that an application time of at least 60 min was necessary to label S-phase cells, a 2% concentration of BrdU was needed to obtain consistent labeling and aqueous vehicles are satisfactory solvents for BrdU preparations. Epidermal keratinocytes and S-phase cells in the upper portion of dermis were clearly labeled after either intraperitoneal injection of BrdU or after administration by means of the OD method. To ascertain whether this latter method could provide an effective alternative to intraperitoneal injection, we compared the labeling patterns of both methods with respect to the speed of migration of BrdU-labeled basal cells from the basal layer to the horny layer of epidermis. Using either of these two methods, basal keratinocytes were labeled immediately after administration. Three days after the first administration, BrdU-labeled cells were detected in the middle layer of the epidermis, but after 8 days, they were no longer evident in epidermal tissue. As another means of comparing both methods, we used antibody to proliferating cell nuclear antigen (PCNA) and compared the ratio of PCNA-positive basal cells to BrdU-labeled basal cells. The number of PCNA-positive cells was about 4.6 times greater than the number of BrdU-labeled basal cells by both methods. We concluded that the OD method could be used as a substitute for intraperitoneal injection in order to observe cell kinetics using bromodeoxyuridine.

The ontogeny of sulphated glycoconjugate-producing cells in the rat fundic gland has been studied using high iron diamine (HID), Alcian Blue (AB) at pH 1.0, high iron diamine in combination with Alcian Blue at pH 2.5 (HID-AB), cationic colloidal gold (CCG) at pH 1.0 under light microscopy and CCG (1.0), HID-thiocarbohydrazide (TCH)-silver proteinate (SP)-physical development (PD) under electron microscopy. From day 19.5 of gestation, sulphated glycoconjugate-producing cells were discernible under both light and electron microscopy. The development of such cells can be classified into four stages: (1) a prenatal period from day 19.5 of gestation extending to 0.5 days after birth; (2) 1 day to 2 weeks after birth; (3) 2 to 4 weeks after birth; and (4) the final period from 4 to 8 weeks after birth. Glycoconjugate-producing cells reached maturity by 4 weeks after birth. Our results indicated that glycoconjugate-producing cells were cells along the wall of foveolar lumen, but not those covering the gastric mucosa surface. Our results also suggested that the trans to transmost Golgi apparatus lamellae were the sites of sulphation in the developing rat stomach.

Trichosanthesjaponica agglutinin I (TJA-I) having a high affinity to the NeuAcα2-6Galβ1-4GIcNAc terminal sequence was used for an immunohistochemical study to analyze the expression of the NeuAcα2-6Gal structure in human colonic tissues. A TJA-I binding substance was detected in the mucosa of colonic carcinoma, but not in the normal and transitional mucosa. The TJA-I staining was correlated with the degree of histo logical differentiation of the carcinoma. TJA-I preferentially stained well-differentiated and moderately differentiated adenocarcinoma. In the carcinoma containing mucin lakes, the TJA-I staining reaction was either weak in intensity or negative. A comparative study using monoclonal antibody against sialosyl-Tn antigen (NeuAcα2-6-GaINAc) known to be expressed in colonic carcinoma tissues revealed that the areas exhibiting strong expressions of siaosyl-Tn antigen separated from the areas vividly reactive for the TJA-I staining. In addition, the expression of sialosyl-Tn antigen, unlike the TJA-I staining, was strong in the mucinous areas of the carcinoma. The results obtained in the present study suggest the possibility that a comparative staining method with TJA-I and monoclonal antibody against sialosyl-Tn antigen could be helpful to the diagnosis of human colonic carcinoma.

As an alternative method to 3H-thymidine radioautography, immunohistochemical staining against incorporated bromodeoxyuridine (BrdU), an analogue of thymidine, was performed on rat stomach and small intestine organs embedded in Lowicryl K4M resin. After the administration of BrdU to animals intraperitoneally, organ tissues were removed and processed in a routine manner for resin embedding procedures. Semithin and ultrathin serial sections were cut and incubated in mouse anti-BrdU monoclonal antibody and stained with anti-mouse IgG or protein A coupled with gold colloid. Semithin sections were further intensified by physical development using silver acetate as an ion donor. In the electron microscopic labeling, pretreatment with hydrogen peroxide increased the labeling intensity. The findings from both light and electron microscopic observation were compared and a good correlation was obtained. Various labeling patterns were found in S-phase cell nuclei from stomach and small intestines in vivo. Labeling patterns of nuclei matrix were divided into five subtypes according to labeling patterns at the electron microscopic level.

A 21-year-old man with childhood-onset mental retardation, non-obstructive hypertrophic cardiomyopathy, and vacuolar myopathy is presented. A histopathological study of biopsied skeletal muscle showed lysosomal glycogen storage mimicking acid maltase deficiency, but biochemical analysis showed normal acid α-glucosidase activity. Glycogenosomes were also recognized in endothelial cells on electronmicroscopic examination of biopsied skeletal muscle. Magnetic resonance imaging (MRI) findings in the head revealed the involvement of the central nervous system. This is a new type of lysosomal glycogen storage disease with multisystemic involvement. The specific biochemical defect in this disorder remains to be elucidated.

We prepared a monoclonal antibody (MAb) against N-acetylglucosaminide beta 1----4 galactosyltransferase purified from F9 embryonal carcinoma cells. The MAb recognized the protein portion of the enzyme, since it inhibited galactosyltransferase activity, reacted with the enzyme both from F9 cells and from bovine milk, and did not exhibit anti-carbohydrate activity. Using this MAb, we studied the subcellular localization of the enzyme by immunoelectron microscopy. Intense staining was observed in trans-Golgi stacks within testicular interstitial cells and mucous neck cells, confirming the specificity of the immunological reaction. Cell surface galactosyltransferase was detected in the following regions: cultured cells such as F9 embryonal carcinoma cells, testicular interstitial cells, seminiferous tubule epithelial cells, Sertoli cells, the head of the epididymal sperm, epididymal epithelial cells, and apical surfaces of epithelial cells in the fundic gland and of intestinal goblet cells. The use of Triton X-100 intensified the cell surface immunoreactivity, and in certain cases the mode of distribution of the cell surface enzyme was different from that described in previous reports. In addition, nuclear envelopes of cultured cells were distinctly stained. The possible significance of the latter finding is discussed in relation to recent advances in nuclear localization of glycoproteins.

Lectin histochemical studies were performed on frozen sections of biopsied skeletal muscle from two unrelated patients with lysosomal glycogen storage disease without acid maltase deficiency. The clinical manifestations of these patients, myopathological features and biochemical characteristics are similar to those first reported by Danon et al. in 1981. Routine myopathological findings showed vacuolar myopathy and excess of glycogen with or without glycogenosomes resembling acid maltase deficiency (AMD), but the biochemical activity of acid α-glucosidase was normal.Ten lectins with varying sugar specificities were used as probes. The results were compared with normal muscle and with one patient who had the typical manifestations of adult onset AMD. Though myofibers themselves were never stained in normal muscle, positively stained myofibers with or without vacuolations were found in diseased muscle. Vacuolar membranes and their contents were strongly stained by almost all the lectins applied. Ulex europaeus agglutinin-I (UEA-I) specific to L-fucose stained the perimeter of the disturbed muscle in addition to the vacuoles.The staining pattern of the vacuolations and perimeter of disturbed myofibers was different from that found in AMD. In AMD, Triticum vulgaris agglutinin (WGA) and Limax flavus aggutinin (LFA) faintly stained the vacuolar membranes and contents. These preliminary investigations of abnormal lectin staining patterns may provide a key to understanding the pathomechanism of this unique lysosomal glycogen storage disease.

Proliferation, apoptosis and p53 protein expression in adult T-cell leukemia (ATL) cells were investigated. Twenty peripheral blood tissue specimens (PBTS) comprising 7 cases of acute type ATL, 7 cases of chronic type ATL and 6 other leukemias were examined by means of antigen retrieval and the polymer method employing anti-Ki67 antigen (MIB-1), anti-cleaved caspase-3, anti-single stranded DNA and three kinds of anti-p53 protein antibodies including DO7. Most acute and chronic cases of ATL included more than 10% MIB-1-positive proliferating leukemia cells and more than 1% cleaved caspase-3-positive apoptotic cells. Some cells which were positive for both MIB-1 and anti-cleaved caspase-3 antibody were observed in acute type ATL. Nuclear deposition of p53 protein labeled by DO7 was often found in acute type (p < 0.05). Within the medium-sized population of ATL cell nuclei, DO7-positive ATL cells had a smaller nuclear area factor (long axis x short axis) than DO7-negative ATL cells. A few proliferating ATL cells entered apoptosis, and the appearance of a subclone of ATL cells with nuclear deposition of p53 protein labeled by DO7 characterized acute type.

A newly identified gene MK is transiently expressed in early stages of retinoic acid-induced differentiation of embryonal carcinoma cells (Kadomatsu, K., M. Tomomura, and T. Muramatsu, 1988. Biochem. Biophys. Res. Commun. 151:1312-1318). MK gene has been predicted to code a polypeptide that is rich in basic amino acids and cysteine and is not related to any other peptides so far reported. In the present study, we investigated MK expression during mouse embryogenesis by in situ hybridization. The MK transcript was detected all over the embryo proper of the 7-d embryo, while it was not detectable in the 5-d embryo. The ubiquitous expression continued in the 9-d embryo proper. On the 11th-13th d of gestation, the sites where MK gene was intensely expressed became progressively restricted; these sites were the brain ectoderm around the lens and brain ventricles, the anterior lobe of the pituitary gland, the upper and lower jaw, the caudal sclerotomic half of vertebral column, the limbs, the stomach, and the epithelial tissues of the lung, the pancreas, the small intestine, and the metanephros. These areas include the region where secondary embryonic induction is prominent. In the 15-d embryo, only the kidney expressed MK significantly. These data suggest that MK gene plays a fundamental role in the differentiation of a wide variety of cells; MK gene may also play some specific roles in generation of epithelial tissues, and remodeling of mesoderm.

The development of Wistar rat fundic glands was studied using Griffonia simplicifolia agglutinin-IB4 (GSA-IB4) at the light microscopic and electron microscopic levels.In adult rats, GSA-IB4 specifically labeled the intracellular canaliculi and the apical surface on the fundic gland parietal cells, whereas the other sites of these cells were very weakly or rarely labeled.Ontogenic studies revealed that at 18 days of gestation, GSA-IB4 positive reaction was found along the whole cell surface of the cells on the fundic gland and immature intracellular canaliculi of the primitive parietal cells. At 1 day after birth, the positive reaction was restricted to the parietal cells. GSA-IB4 specifically labeled the intracellular canaliculi and the apical cell surface of the parietal cells, but the labeling of the basolateral cell membrane of parietal cells decreased. At the point of weaning, 3 weeks after birth, GSA-IB4 strongly labeled the intracellular canaliculi whereas the basolateral membrane was weakly labeled. This staining pattern did not change basically until adult stage.From these results, it is concluded that the functional maturation of the parietal cells may occur at weaning in spite of their early appearance of morphological characters.

While intramuscular wire electrodes (IWE) for the measurement of neuromuscular function offer high spatial resolution for examining single motor unit activity, the resulting damage to muscle tissue and mechanical instability should be considered. We examined the influence of IWE type and component parts on muscle damage using light microscopy in rats and confirmed that intramuscular pressure influences the mechanical stability of IWE. Three types of electrode, coiled electrodes with or without suture material inside and a straight electrode, were inserted into the soleus, gastrocnemius and tibialis anterior muscles. Transverse serial sections (5 microm) of these muscles in the vicinity of the electrodes were stained with haematoxylin and eosin. Less structural damage was observed in the vicinity of the recording points (leading-off surface; 50 microm diameter) for all electrode types compared to the electrode body. No differences in the extent of tissue damage were observed around the recording points for all electrodes. However, compared to straight electrodes, the extent of damaged tissue around the bodies of coiled electrodes was significantly (P0.0001) greater. The average distance between the recording points and the electrode body was1 mm for all electrodes. Intramuscular pressure at rest and maximal twitch contraction were 1.1 +/- 0.5 and 49.4 +/- 4.0 mmHg, respectively. Coiled IWEs became well integrated with muscle fibres, stabilizing electrode localization and facilitating electromyographic recordings without causing significant muscle damage.

The aim of this study was to investigate cell kinetics and ultrastructural changes during carcinogenesis using a hamster 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced tongue cancer model. Five squamous cell carcinomas, five dysplastic epithelia, seven hyperplastic epithelia, and four normal epithelia were obtained from 21 hamster tongues by applying 1.0% acetone solution of DMBA on the left lingual mucosa after scratching with a root canal broach. Ultrastructural examination revealed that the number of microvilli increased, whereas that of desmosomes decreased during carcinogenesis. Cell proliferation was analyzed by means of 5-bromodeoxyuridine (BrdU) immunohistochemistry and in situ hybridization (ISH) for histone H3 mRNA. The BrdU and histone H3 mRNA labeling indices (LIs) were lowest for normal epithelium, higher for hyperplastic and dysplastic epithelia, and highest for squamous cell carcinoma. Cytoplasmic histone H3 mRNA and nuclear BrdU were localized in virtually identical areas of serial sections. The correlation coefficient for the relationship between these two LIs was 0.97 (P ≪ 0.001). These results suggest that the assessment of cell proliferation using H3 mRNA ISH will be a useful technique for investigating biological behavior during carcinogenesis.

Basigin is a glycosylated transmembrane protein belonging to the immunoglobulin superfamily. It is thought to play roles in intercellular recognition involved in cell differentiation. We previously demonstrated at the light microscope level a correlation between basigin expression and epidermal differentiation. In the present study, the ultrastructural localization of basigin in normal human epidermal keratinocytes was investigated by immunoelectron microscopy. The basigin labeling was strongest on membranes of basal cells, weaker on prickle cells, and absent in granular and horny cells. On the membrane of basal cells, labeling was observed on the apical and lateral sides but not on the dermal side. Gold particles were mostly observed on the surface of microvilli, especially on their tips. There were fewer on the intermicrovillous membrane and they were absent on the desmosome. These results are consistent with our previous report that basigin expression is correlated with differentiation of epidermal keratinocytes. Microvilli on basal and suprabasal keratinocytes might play roles in the differentiation of keratinocytes through basigin on the tips of microvilli.

Class III mucin, identified by paradoxical concanavalin A staining, is confined to gastric gland mucous cells and is an essential component of the gastric surface mucous gel layer. The pretreatment required has hampered the application of this method to electron microscopic studies. Antibody HIK1083 reacts selectively with class III mucins. The present study was undertaken to explore, electron microscopically, the immunoreactivity of the human stomach to HIK1083. We examined normal mucosa from resected human stomachs (five cases; formalin-fixed, paraffin-embedded) and gastric biopsy specimens from patients with early gastric cancer [nine cases; glutaraldehyde- and osmium-fixed, epoxy-embedded (seven cases) and half-strength Karnovsky's solution-fixed, Lowicryl K4M-embedded (two cases)]. Immunostaining with HIK1083 and anti-lysozyme antibody was examined under light and electron microscopes. Gland mucous cells were labeled with HIK1083, and lysozyme was detected in some gland mucous cells and surface mucous cells. Electron microscopically, the secretory granules of gland mucous cells contained a single electron-dense core. HIK1083-positive mucins and lysozyme coexisted in the secretory granules of gastric gland mucous cells. HIK1083-reactive mucins and lysozyme were distributed in the matrix and in the dense core of these secretory granules, respectively. HIK1083 can be used for electron immunohistochemistry.

Fabry disease is an X-linked inborn error of glycosphingolipid catabolism resulting from a deficiency of lysosomal alpha-galactosidase activity. Globotriaosylceramide accumulates predominantly in lysosomes of various tissues. Former studies have clarified the nature of this disease, and the accumulated materials in the lysosomes have been analyzed using biochemical techniques. In the present study, transmission electron microscopy was used to reveal the fine structure of these lysosomal deposits, and sugar residues in the lysosomal deposits in Fabry disease were examined by lectin histochemistry combined with enzyme digestion. This is the first report to describe the lysosomal sugar residues in Fabry disease analyzed using lectin histochemistry at the ultrastructural level. With these techniques, we were able to detect alpha-galactosyl, beta-galactosyl and glucosyl sugar residues in the lysosomal deposits. The experimental procedures used in this study have considerable potential for use in investigations of glycolipid and glycoprotein storage diseases without the need for complex methodology and expensive materials.

The ontogeny of pepsinogen C-producing cells in rat fundic glands was studied by means of light and electron microscopy using an antiserum raised against a synthetic peptide based on rat pepsinogen C. To confirm the immunocytochemistry results, the expression of rat pepsinogen C messenger RNA (mRNA) in the fundic gland was also examined by in situ hybridization using a digoxigenin-labeled RNA probe. In adult rats, pepsinogen C was produced by chief cells, mucous neck cells, and intermediate mucopeptic cells. Pepsinogen C-producing cells appeared in embryos as early as 18.5 days’ gestation. The development of these cells could be classified into four stages: (1) 18.5 days’ gestation to 0.5 days after birth; (2) 0.5 days to 2 weeks after birth; (3) 3–4 weeks after birth; (4) 4–8 weeks after birth. In embryos and young animals, pepsinogen C-producing cells were mucopeptic cells. By 4 weeks after birth, mucous neck cells could be distinguished morphologically. The maturation stages of the chief cells could be traced by electron microscopy along the longitudinal axis of the rat fundic gland by double-staining with anti-pepsinogen C antibody and periodic acid-thiocarbohydrazide-silver proteinate. Positive reactions for pepsinogen C and pepsinogen C mRNA expression were detected in mucous neck cells. Therefore, we conclude that mucous neck cells are precursor cells of chief cells. Mucous neck cells, intermediate cells, and chief cells are in the same differentiating cell lineage.

Background : Platelet-derived endothelial cell growth factor (PD-ECGF) is known to promote the development of new blood vessels, which are fundamental to tumor growth and metastasis. We previously found that thymidine phosphorylase (dThdPase) and PD-ECGF are the same protein. Purpose : We retrospectively examined the expression of dThdPase in primary colorectal carcinomas, its association with angiogenesis and clinicopathologic findings, and its prognostic value. Methods : Tissues were obtained from the tumors of 163 patients whose colorectal carcinomas were completely removed by surgery. Microvessels assessed by immunostaining endothelial cells for factor VIII were counted on a 400x field in the most active areas of neovascularization within the tumor. We purified the monoclonal antibody against dThdPase and studied the expression of dThdPase in the same serial sections used for the detection of factor VIII. Those who carried out microvessel counting and dThdPase expression assessment had no knowledge of clinicopathologic findings. The significance of dThdPase in the prognosis of patients with colorectal carcinomas was also examined in the survival analysis of mortality follow-up data covering the period between 1984 through 1991. Reported P values are from two-sided tests of statistical significance. Results : The mean microvessel count (± standard deviation) in dThdPase-positive colorectal carcinoma specimens (17.5 ± 7.2) was higher (P

Type I receptors for bone morphogenetic proteins (BMPs), i.e., BMPR-IA and BMPR-IB, are transmembrane serine/threonine kinases, that bind osteogenic protein-1 (OP-1, also termed BMP-7) and BMP-4. Using antibodies specific to BMPR-IA and -IB, we have studied the expression of BMP type I receptors in the bone formation process during embryonic development and fracture healing. In the mouse embryo, both BMPR-IA and -IB were expressed in condensing mesenchymal cells at 13.5 days post coitum (p.c.). At 15.5 days p.c., expression of BMPR-IB, but not of BMPR-IA, was observed in the cells in perichondrium of developing cartilage. At 17.5 and 19.5 days p.c., expression of both receptors was observed in chondrocytes and in osteoblasts. In normal rat adult bone, expression of BMPR-IA, but not of BMPR-IB, was observed in osteoblasts in the periosteum. Three days after the femoral fracture, expression of BMPR-IA and -IB was up-regulated in cells at the proliferating osteogenic layer of the periosteum. On day 7, both receptors were found in fibroblast-like spindle cells and chondrocytes in the endochondral ossification sites, and osteoblasts in the newly formed trabecular bone. Expression of BMPR-IA was higher than that of BMPR-IB in osteogenic layer on day 3 and in osteoblasts in the trabecular bone on day 7. On day 14, expression of BMP type I receptors was observed at similar sites, albeit with lower expression levels than were observed on day 7. The present data suggest that expression of BMP type I receptors is up-regulated during bone formation, and that they may play important roles in bone morphogenesis.

We investigated the ultrastructure of blood group antigens A, B, and H in human eccrine glands by means of the immunogold labeling technique. Blood group antigens A, B, and H were found in the Golgi apparatus, secretory granules, and over the apical and basolateral cell membranes of dark cells of eccrine glands depending on the blood group phenotype of the donors. Both A and B antigens were found in the dark cells of AB donors. The labeling pattern of the Golgi stacks seemed to have a polarity whereby the anti-blood group A antibody labeled all the stacks, whereas anti-blood groups B and H bound to the trans side of the Golgi complex. These observations suggest that the blood group substances are secreted into the lumen after being processed through the Golgi apparatus and the immature and mature granules in the dark cells of human eccrine glands.

Mucous glycoproteins of rat Brunner's gland were examined ultracytochemically to elucidate the intracellular localization and sites of glycosylation in relation to the functional polarity of the cell organellae using a combination of hydrophilic resin embedment and postembedding staining with labeled lectins. A few cis cisternae were stained with HPA (Helix pomatia agglutinin, specific for terminal alpha-N-acetylgalactosamine) and DBA (Dolichos biflorus aggulutinin, specific for terminal alpha-N-acetylgalactosamine). Trans cisternae as well as cisternae following secretory granules were stained with HPA and RCA-I (Recinus communis agglutinin I, specific for terminal beta-galactose). UEA-I (Ulex europaeus agglutinin I, specific for terminal alpha-L-fucose) seemed to bind to transmost cisternae rather than trans cisternae. HPA bound to the whole cisternae, but to the cis or trans cisternae depending on a cell type. Apical mucous granules were stained with UEA-I, RCA-I, and HPA, but not with DBA. DGA seemed to be a good marker of the cis side of the Golgi apparatus and cell membrane. The glycosylation of mucous glycoprotein in rat Brunner's gland was partly clarified electron microscopically.

The localization of lectin-binding sites in the rat gastric mucosa was investigated using horseradish peroxidase-labeled lectin conjugates (Con A, DBA, PNA, RCA-1, SBA, UEA-1, WGA). The glandular epithelium of the cardiac gland was clearly stained with DBA, PNA and SBA. Surface epithelium of the fundic glands was intensely labeled with SBA and UEA-1, but weakly with RCA-1, and WGA. Staining for foveolar cells was intense with SBA and moderate with PNA, while Con A and RCA-1 showed negligible reactivity. Mucous neck cells were intensely stained with SBA, moderately with PNA, weakly with Con A, RCA-1 and WGA, and only, faintly with DBA. Chief cells reacted positively only with WGA. Surface epithelium of the pyloric gland was moderately labeled with DBA, SBA and WGA, and faintly with the other lectins. Glandular epithelial cells, which revealed a similar pattern to that of the surface epithelium, were stained more intensely. The present study provides additional information for elucidating the cellular and regional differences in lectin binding to the rat gastric mucosa.

The fundic gland of the rat stomach was studied using the low-temperature embedding resin Lowicryl K4M and postembedding staining with lectin/colloidal-gold (CG) conjugates. Intense labeling with Ricinus communis agglutinin I was observed not only in mucous-producing cells but also in parietal cells. In contrast, Helix pomatia agglutinin (HPA) only labeled mucous neck cells and intermediate cells between mucous neck cells and chief cells. The other epithelial cells present in the rat fundic gland showed virtually no reaction with this lectin. Our results indicate that HPA might be a marker lectin of mucous neck cells and their derivatives. The combination of embedding in the hydrophilic resin Lowicryl K4M and postembedding staining with lectin-CG conjugates provided satisfactory staining results, and made it possible to visualize the precise distribution of terminal glycoconjugates in intracellular components as well as on the plasma membrane.

Postembedding staining of intracellular carbohydrates of rat Brunner's gland cells embedded in Epon and acrylamide was carried out with Ricinus communis agglutinin-ferritin, concanavalin A-ferritin, and wheat germ agglutinin-ferritin conjugates. Th Golgi vacuoles and mucous granules were stained with these conjugates. In each staining, the tissues embedded in acrylamide were stained more strongly than those embedded in Epon. The staining intensity of wheat germ agglutinin-ferritin was the strongest among the three conjugates and the staining intensity of Ricinus communis agglutinin-ferritin was stronger than that of concanavalin A-ferritin in both embedding methods. Free ferritin showed almost no binding to these structures and staining with the conjugates was inhibited by the addition of appropriate competitive sugars to the staining solutions. Osmium-postfixed tissues were not stained well with the conjugates. Washing of the sections with bovine serum albumin solution after staining was an essential step in the present method to reduce the nonspecific adsorption of the conjugates. The present method was very simple and had good reproducibility.

The localization of pepsinogen in rat stomachs was investigated by a postembedding immunoferritin method. When the preparations embedded in Epon were used, the secretory granules of chief cells were stained heavily and the granules of mucous neck cells were stained moderately. The secretory granules of cells intermediate between mucous neck cells and chief cells showed a bizonal staining; the electron dense parts were stained heavily and the electron lucent parts were stained moderately. The secretory granules of pyloric gland cells, on the other hand, were labeled faintly. However, the secretory granules of surface mucous cells, foveolar mucous cells, endocrine cells, cardiac mucous cells and cardiac serous cells were not stained by the method. The protein A-gold method showed a similar staining pattern of pepsinogen to that of the immunoferritin method. When the samples embedded in Lowicryl K4M were used to enhance the stainability of pepsinogen, essentially the same staining pattern as that of the samples embedded in Epon was obtained. In addition, the Golgi apparatus and the rough surfaced endoplasmic reticulum were more easily stained.

Rabbit bone marrow cells have been studied by means of light- and electron-microscopic radioautography and X-ray microanalysis. Carrier free sulfuric acid was used as the radioactive precursor in this experiment. Immature granulocytes showed more active incorporation than mature ones. Silver grains were observed in the Golgi apparatus and granules in three kinds of granulocytes. Electron-microscopically, immature granules showed the incorporation of inorganic sulfate, while mature ones did not. Sulfur was detected in all kinds of granules of the three granulocyte types by X-ray microanalysis. It is concluded that incorporated inorganic sulfur may be utilized for the synthesis of acid glycosaminoglycans and the sulfur detected by X-ray microanalysis may be that contained in the acid glycosaminoglycan. The sulfur detected in the specific granules of the heterophil probably derives from proteins or polypeptides incorporating sulfur containing amino acids.

The histochemical feature of epithelial mucins of the rat proximal colon differed from that of the distals. The regional differences were elucidated by various mucin-staining methods, such as periodic acid-Schiff (PAS), Alcian blue (pH 2.5) -PAS (AB-PAS), high iron diamine (HID) -AB (pH 2.5), and O-acylated sialic acid specific staining: periodic acid-thionin Schiff (PATS) /-periodate-borohydride technique (PBT) /potassium hydroxide (KOH) /periodic acid-Schiff (PAS) technique for light microscopy, periodic acid (PA) -thiocarbo-hydrazide (TCH) -silver proteinate (SP) staining, O-acylated sialic acid specific staining and dialyzed iron staining for electron microscopy. The distribution of sugar residues of the glycoproteins in the colonic epithelial mucin was also examined by lectin-horseradish peroxidase (HRP) staining method. Goblet cells at the upper half of the crypt in the proximal colon contained mainly neutral mucin and at the lower half, their mucin showed alcianophilic nature predominantly. Sulfomucin secreting-cells were distributing only at the near luminal surface of the proximal colon and all through the crypt of the distal colon. Some goblet cells, columnar cells and vacuolated cells contained O-acylated sialoglycoconjugates. The lectin staining-patterns differed from each other. Some lectins stained the upper half goblet cell-mucin only or the lower half goblet cell-mucin, while other lectins stained goblet cells of the whole crypt. It seemed that the histochemical staining features changed at the branching site of the crypt in the proximal colon. On the other hand, such a transitional zone was obscure in the distal colon.