Search

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.

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.

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.

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.

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

Using a high electron resolution staining method, cationic colloidal gold (CCG, pH 1.0) staining, we studied the fine structural localization of sulfated glycosaminoglycans (GAGs) in various maturational stages of guinea pig neutrophils. Azurophil and specific granules of neutrophils reacted positively to CCG, with variety in labeling according to maturation. All immature azurophil and specific granules were labeled selectively. Mature granules lost their affinity with CCG. CCG-positive labeling was also observed in the trans to trans-most Golgi apparatus of promyelocytes and myelocytes. Prior absorption with poly-L-lysine prevented CCG labeling of tissue sections. Mild methylation of ultrathin sections at 37C did not alter CCG labeling, whereas CCG labeling disappeared after active methylation at 60C. Treatment with chondroitinase ABC or heparinase I abolished the majority of CCG labeling. These findings suggest the existence of sulfated GAGs not only in immature azurophil but also in immature specific granules of neutrophils. Sulfation of GAGs occurs in the trans- to trans-most Golgi apparatus of neutrophil granulocytes. A possible correlation between accumulation of sulfated GAGs and maturation of specific granules in neutrophils is also discussed.

We examined the presence of sialyl glycoconjugates in specific granules from murine bone marrow eosinophils. Lectin cytochemistry using Maackia amurensis lectin II (MAL II) specific for sialyl alpha-2,3 galactose residues demonstrated positive labeling in both immature and mature specific granules. Pretreatment with Clostridium neuraminidase or keratanase II eliminated the positive labeling of MAL II in the specific granules. High iron diamine-thiocarbohydrazide-silver proteinate physical development (HID-TCH-SP-PD) staining, which is specific for sulfated glycoconjugates, also positively labeled immature specific granules lacking crystalloids but not mature granules with crystalloids. Pretreatment with a combination of chondroitinase ABC and keratanase, or a combination of chondroitinase ABC and keratanase II, eliminated the positive labeling obtained with HID-TCH-SP-PD. These results indicate that the sialyl residues detected by MAL II are expressed as terminal sugar residues of keratan sulfate proteoglycan, which appears to be of the corneal type in view of its sensitivity to keratanase and keratanase II. (J Histochem Cytochem 47:481-488, 1999)

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.

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.

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.

Sites of glycosylation were investigated using postembedding and preembedding lectin staining. Positive labeling in the luminal side of perinuclear spaces and endoplasmic reticulum was observed by N-acetylgalactosamine and mannose binding lectins. Cis lamellae of the Golgi apparatus were stained by N-acetylgalactosamine and mannose binding lectins. Medial to trans lamellae were mainly labeled by galactose binding lectin and transmost lamellae were labeled by sialic acid and fucose binding lectins. The present results elucidated the route of glycoconjugate synthesis in relation to cell organelle.

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.

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.

We examined the intracellular localization of sugar residues of the rat gastric surface mucous cells in relation to the functional polarity of the cell organellae using preembedding method with several lectins. In the surface mucous cells, the nuclear envelope and rough endoplasmic reticulum (rER) and cis cisternae of the Golgi stacks were intensely stained with Maclura pomifera (MPA), which is specific to alpha-Gal and GalNAc residues. In the Golgi apparatus, one or two cis side cisternae were stained with MPA and Dolichos biflorus (DBA) which is specific to terminal alpha-N-acetylgalactosamine residues, while the intermediate lamellae were intensely labeled with Arachis hypogaea (PNA) which is specific to Gal beta 1,3 GalNAc. Cisternae of the trans Golgi region were also stained with MPA, Ricinus communis I (RCA I) which is specific to beta-Gal and Limax flavus (LFA) which is specific to alpha-NeuAc. Immature mucous granules which are contiguous with the trans Golgi lamellae were weakly stained with RCA I, while LFA stained both immature and mature granules. The differences between each lectin's reactivity in the rough endoplasmic reticulum, in each compartment of the Golgi lamellae and in the secretory granules suggest that there are compositional and structural differences between the glycoconjugates in the respective cell organellae, reflecting the various processes of glycosylation in the gastric surface mucous cells.

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.

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.

We investigated non-specific staining in a catalyzed reporter deposition (CARD) reaction and improved its blocking methods in supersensitive immunohistochemistry, based on our simplified catalyzed signal amplification (CSA) system (Hasui et al. 2002). In the CARD reaction using biotinyl tyramide, non-specific staining could be reduced by pretreatment with a casein solution or 3% bovine serum albumin (BSA)-phosphate buffer saline (PBS) with 0.1% Tween 20. In the CARD reaction using FITC-labeled tyramide, non-specific staining could be blocked by pretreatment with 0.3% BSA-PBS with 0.1% Tween 20 or 3% polyethylene glycol-PBS with 01% Tween 20. Thus, our new simplified CSA system features: 1) destruction of the endogenous peroxidase activity; 2) blocking of the nonspecific reaction of the primary antibody; 3) a primary antibody reaction; 4) blocking of the non-specific reaction of the polymer reagent by casein treatment; 5) a polymer reaction; 6) blocking of the non-specific reaction of CARD reaction by casein treatment; 7) a CARD reaction; and 8) detection of deposited tyramide. This new system proved useful for detecting an extremely low amount of antigen in the endogenous biotin-rich tissues such as the gastrointestinal tract and liver. By this method, the Ki67 antigen in the G1 phase cell cycle could be detected and a metabolic disorder of the Ki67 antigen was implicated in a carcinoid tumor in the stomach. We believe that this new simplified CSA system represents a new standard of supersensitive immunohistochemistry for use in light-microscopic investigation.

Double autoimmunostaining by a sequential twice-repeated enzyme-labeled polymer method was examined on archival paraffin sections of formalin-fixed human tissue using an autoimmunostaining apparatus to determine optimal conditions for glycine treatment, to select the best combination of dyes for the horseradish peroxidase–hydrogen peroxide reaction, and to investigate mounting methods for preparing permanent specimens. The optimal glycine treatment determined by changing the incubation time in 0.1 M glycine hydrochloride buffer, pH 2.2, was glycine buffer washing three times for 1 min each, with suppression of nonspecific binding of the primary antibody by protein blocking. Combinations of DAB and AEC, SG and AEC with Ultramount, and DAB and VIP or No-vaRED and SG with the VectaMount were found usable for the double autoimmunostaining, based on color analysis of the dyes. Pairs of primary antibodies, CD68 and anti-fascin antibodies CD3 and CD79a, and anti-Ki-67 antigen and anti-p53 antibodies were applicable in double autoimmunostaining with appropriate antigen retrieval for each pair of primary antibodies. Consequently, good sequential double autoimmunostaining should include masking the nonspecific binding of primary antibodies, optimal glycine treatment, and selection of adequate dyes and mounting methods.

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.

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.

The acidic glycoconjugates of mouse ileum Paneth cells were examined with the aid of light and electron microscopy, using cationic colloidal gold (CCG) as a probe. Specimens of mouse ilea were fixed in half-strength Karnovsky's fixative and embedded in Lowicryl K4M resin. Semithin and ultrathin sections were cut of examination with light and electron microscopy, respectively. Examination of the sections using light microscopy revealed the positive staining of CCG at pH 1.0 and pH 2.5, which was detected at the rim of secretory granules and at the supranuclear regions of the Paneth cells. At pH 4.0, in addition to staining of the secretory granule rim, weak staining was observed in the granule core. At pH 7.2, the cytoplasm other than secretory granules exhibited positive CCG staining. Examination of the sections using electron microscopy, at pH 1.0, the trans lamellae of the Golgi apparatus, the rim of the secretory granules, and lysosomes were labeled selectively by CCG. At pH 2.5, labeling was also discernible over the same structures in the cells. However, at this pH, the labeling intensity was stronger than that at pH 1.0, due to the dual labeling of sulfated and sialylated glycoconjugates in these structures. At pH 4.0, the Golgi apparatus, rims and cores of secretory granules and ribosomes were labeled. Lysosomes and nuclei were also positively stained. At pH 7.2, the rims of secretory granules were not stained. The present results indicate that the CCG method gives good resolution and contrast when applied to staining, and therefore is useful for the specific staining of glycoconjugates such as sulfated, sialylated and phosphated glycoconjugates for light and electron microscopy.

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.

The mucous glycoproteins of mucous cells in the lower half of the rat proximal colon were examined ultracytochemically to study the intracellular sites of glycosylation in relation to the cell differentiation and the functional polarity of the cell organellae using a combination of a hydrophilic resin embedment and a postembedding staining technique with lectin gold com plexes.In the immature mucous cells located near the branching point of the glandular crypt, the Golgi stacks and mucous granules were stained with soybean agglutinin (SBA), Ricinus communis agglutinin-I (RCA-I) and Helix pomatia agglutinin (HPA). In the mature mucous cells at the bottom of the crypt, the cis region of the Golgi stack was stained with SBA, a few intermediate saccules were stained with RCA-I, and both the cis and trans sides of the cisternae and mucous granules, but not the intermediate lamellae, were stained with HPA. After neuraminidase digestion of ultrathin sections, the RCA-I binding sites were greatly increased. Ulex europeus agglutinin-I (UEA-I) and Limax flavus agglutinin (LFA) positively stained the trans side lamellae and mucous granules of the mucous cell at the bottom of the glandular crypt.The processing of the glycosylation of the mucous glycoprotein in the lower half crypt of the rat proximal colon 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.

The glycogen of megakaryocytes and blood platelets has been investigated in glutaraldehyde and osmium tetroxide fixed tissues by the periodic acid-thiocarbohydrozide-silver proteinate method (PA-TCH-SP). The PA-TCH-SP method involves the staining of intracytoplasmic glycogens more densely than the routine lead citrate method. Glycogen having a mean particle diameter of 21.1 nm has been shown localizing in the matrix of mature megakaryocytes, while that of glycogen in the platelets was 26.2 nm. The staining pattern of the glycogen in blood platelets was classified into three groups according to staining intensity. It is found that the PA-TCH-SP method is a very suitable one for the demonstration of intracytoplasmic glycogen from the viewpoints of reaction specificity, reproducibility, fineness of reaction products, sufficiency of electron density, and experimental cost. This method is also a very useful one for differentiating intracytoplasmic glycogens and ribosomes.

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.

Effects of DEHP (di-2-ethylhexyl phthalate) were studied on renal tubular epithelial cells of rats. The 1%, 2% and 4% (w/w) DEHP administered and control rats were sacrificed at 1, 2 and 4 weeks. The tissues were incubated in alkaline DAB medium and embedded in Epon. The 0.5μm thick sections were observed by light microscopy with an ocular micrometer. The number of peroxisomes was determined for the transected area per 25μm2 in 40 different tubular cytoplasms. The differences in the number of peroxisomes between the experimental and control groups were evaluated from the viewpoint of various responses depending on the duration of administration, dose of DEHP, parts off the nephron and sex. Peroxisomal proliferation in DEHP-treated rats occured at low levels of dosage in a short time. The differences in increase between straight parts and convoluted parts of proximal tubules became the most obvious over a long time. The difference depending on sex was not so apparent. The response of proximal tubular epithelial cells to DEHP is not similar to that induced in liver cells by the same drug. Peroxisomes in renal tubular epithelial cells may play a role in lipid metabolism to a lesser extent than those in liver cells.

A routine procedure for electron microscopic radioautography in combination with phenidon developer and the domestic emulsion, Sakura NR-H2, was developed. Good stable radioautograms have not been obtained by simply developing the emulsion coated grids with phenidon developer at any temperature, but the development of these grids at 18°C for 1 min after 30-45 sec gold latensification in freshly prepared gold thiocyanate gives satisfactory results. Some electron microscopic radioautograms developed by this method are presented. The addition of the anionic surfactant, dioctyl sodium sulphosuccinate, to the emulsion was found to be very effective not only for preventing the burst of emulsion but also in obtaining a uniform spread of the emulsion for the wire loop method. Radioautograms obtained with this method are suitable for quantitative electron microscopic radioautographic studies. Details of this method are introduced.

A simplified method for mass production of electron microscopic specimens for histochemistry without using any special instrument is introduced. This is also useful for a very small amounts of tissues. The method prevents the loss of thick sections during the treatment and it does not produce any nonspecific staining. The wide future application is expected.