The glycoproteins of the intestinal mucosa of five vertebrate species including fishes (Sparus auratus), amphibians (Rana perezi), reptiles (Testudo graeca), birds (Gallus gallus) and mammals (Mesocricetus auratus) were studied by means of methods of lectin histochemistry. Goblet cells of the intestine showed different lectin binding patterns depending upon the location in the villi. Thus, goblet cells located in the intestinal glands presented a different reactivity from that of the goblet cells located at the tip of the villi. Differences in lectin stainings between small and large intestines were more marked in higher than in lower vertebrates. WGA binding sites were the most abundant throughout the intestinal mucosa of the animals studied. Golgi areas of the different cell types studied were frequently reactive for DBA and SBA. The results obtained in the Brunner's glands of the hamster were similar to those obtained in other mammals, suggesting a common mucin composition. In the hamster intestinal glands, Con-A stained the Paneth cells.

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Immunohistochemiccal expression of human epidermal growth factor (hEGF) in human tissues and organs is described by the use of polyclonal anti-hEGF antiserum and monoclonal antibody to hEGF generated by the synthetic gene technique. In the digestive system, salivary gland ducts, centro-acinar cells in the pancreas, fundic glands and pylorus glandular epithelium, Brunner's gland, Paneth cells, and hepatic cells showed a positive hEGF reaction. Bronchial glands indicated positive staining in their serous acinar cells and ducts. Kidney contained abundant staining for hEGF in distal convoluted tubules and in some cells of collecting tubules where polyclonal antiserum was used. The prostate gland showed strong hEGF deposition in its epithelium. Mammary gland epithelium, secretory coil cells of the sweat glands, and endometrial glands indicated a positive reaction. In genitalia, Leydig cells of the testis, and lutein cells of the ovary including those in the ovary during pregnancy, indicated the presence of hEGF. In the endocrine organs, acidophilic cells of the anterior pituitary, some cells in its neurohypophysis, follicular cells of the thyroid, acidophilic cells of the parathyroid glands, and the cells in the fasciculus and reticularis of the adrenal glands showed positive staining.
We also found comparatively intense hEGF staining in the epithelial components of various neoplastic lesions, such as salivary plemorphic adenoma, gastric cancer, pancreatic adenocarcinoma, epithelial tumor cells in Wilms tumor, ovarian adenocarcinoma, adenomatous cells of hypertrophied prostate, seminoma and embryonal carcinoma in the testis, decidual cells of hydatidiform mole, mammary adenocarcinoma, oxyphilic adenoma and follicular adenoma in the thyroid, oxyphilic cells of parathyroid adenoma, compact cells of adrenocortical adenoma, and pheochromocytoma cells.
From the present study, it is evident that immunohistochemically detectable hEGF has a widespread distribution in various tissues and organs, including endocrine organs, as well as in neoplastic cells. hEGF may be synthesized by and secreted from those cells. The biologic role of hEGF as mitogen and modulator in those tissues is discussed.

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Specific helical-structure has been previously demonstrated in the lumen of so-called apical tubules (AT) of several absorbing epithelia (kidney proximal tubule, visceral yolk sac and ductuli efferentes); however, their chemical properties remain unknown.
In this study, several cytochemical procedures were applied at electron-microscopic levels to demonstrate the presence of carbohydrates and proteins in the AT of rat kidney proximal tubule cells. After staining for carbohydrates (periodic acid-thiocarbohydrazide-silver proteinate; PA-TCH-SP reaction), heavy reaction products were observed in the lumen of the AT, and the AT originating from the large endocytic vacuoles. Similar products were also shown at the cell surface coat, lysosomes and small portions of the Golgi apparatus. After staining for proteins (phosphotungstic acid or silicotungstic acid in acidic medium; PTA or STA reaction), the helical-structure in the AT was selectively stained, and the intensity of the staining was greatly reduced by treatment with protease.
These observations indicate that the AT probably contain a high concentration of carbohydrates and the material which forms the luminal specializations is composed mainly of proteins, which are readily digested by a proteolytic enzyme.

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Ischaemia in rat soleus muscles induced with a single high dose of 5-hydroxytryptamine caused a loss of activity of alkaline phosphatase, aminopeptidase A and dipeptidyl peptidase IV from capillaries adjacent to and within the most severe muscle lesions. Three days after the insult about 75% activity had returned to the capillaries, and by 6 days normal levels were restored. Loss and recovery of enzyme activity was paralleled by degeneration and regeneration of the muscle fibres.

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In this investigation, we immunohistochemically studied the presence of cells bearing Ia antigen in the stratified squamous epithelia of tongue, esophagus and skin in mice. The developmental expression of Ia antigen positive cells were quantitatively analyzed and compared with skin, by using the sheet method, from birth to the adult stage. Additionally, these cells were morphologically studied by immunoelectron microscopy.
We observed that in case of skin, the increase in the number of Ia positive dendritic cells (Ia⁺DC) was rapid and reached adult stage after day 6, whereas in the tongue and esophagus it showed a gradual increase and reached adult stage by day 28 of postnatal life. However, the number of Ia antigen bearing cells were fewer in tongue and esophagus than in skin on the day of birth. During the whole period of our observation, the cell density was found to be heaviest in skin, followed by tongue and esophagus respectively. The Ia antigen positive cells showed clear dendritic features, the ultrastructure of which showed intracytoplasmic Birbeck granules in the tongue, esophagus, and skin in adult mice (day 28 of postnatal life).
The Ia⁺DC of skin were reported to be identical to the Langerhans cells. Our observations of dendritic features and intracytoplasmic Birbeck granules in the Ia⁺DC of tongue and esophagus suggest that these cells were also Langerhans cells or Langerhans cell-like cells.

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Light and electron microscopic immunocytochemical observations of catechol estrogens in the median eminence of the rat were made using a specific antibody raised against 2-hydroxyestrone coupled to bovine serum albumin and the peroxidase-antiperoxidase technique. Immunoreactive deposits were found in the tanycytes and glia cells. The terminal processes of the reactive cells ended in the perivascular space. Catechol estrogen can cause elevation and/or suppression of serum levels of luteinizing hormone (LH). Thus, our results suggest that catechol estrogens in the reactive cells may be involved in the regulation of secretion of luteinizing hormone releasing hormone (LHRH) in the median eminence.

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The binding and internalization of growth hormone (GH), B-cell growth factor-1linterleukin-4 (BSF-1/IL-4), and Thy-1.2, in the mouse splenic lymphocytes have been described using an electron microscopic immunocytochemical technique. B-cell and T-cell lines in culture are used for the specific binding of BSF-1/IL-4 and Thy-1.2 respectively. A myeloma cell line, X63/Ag 8.653, showed negative bindings for both BSF-1/IL-4 and Thy-1.2. GH binding is found on the surface and in the cytoplasm of most lymphocytes, but is not internalized by the myeloma cells. BSF-1/IL-4 bindings recognized by a monoclonal rat anti-BSF-1/IL-4 antibody (11B11) are found mainly on B-cells, whereas weak bindings are observed on T-cells. Thy-1.2 bindings are specific for T-cells. BSF-1/IL-4 and Thy-1.2 bindings immunolabelled at freezing temperature and reincubation at 37°C for 20min are internalized by B-and T-cells respectively. These results indicate that immunocytochemical technique is a sensitive and powerful tool for detection of specific antigens on the cell surface or in the cytoplasm of cells in the immune system. The internalization of GH, growth factors, and specific antigens presents a possible mechanism of the growth promoting functions in the immune system.

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Taking γ-glutamyl transpeptidase (γ-GT)-monoclonal anti γ-GT system as an example, the effect of microwave fixation for the immunohistochemical study was examined in rat kidney, pancreas, duodenum and liver, using a domestic microwave oven. The results were compared with that of the previous study using conventional fixatives. Best results were obtained in tissues irradiated in 4%-8% paraformaldehyde containing 10% glucose or 5%-10% polyethylene glycol 2000 at 45°-50°C. But, the immunoreaction was not demonstratable along the brush border of the duodenum. Though the microwave fixation is a useful tool for immunohistochemistry, it is necessary to adjust the condition of irradiation and the composition of fixatives to keep the physiological state of each organ or tissue well preserved and for the antigen to stay as it is originally located without loosing its antigenicity.

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PCNA/cyclin was originally described in proliferating mammalian cells as a nuclear protein with an apparent molecular weight of 33, 000-36, 000, and recently found to be a DNA polymerase-delta auxiliary protein. When the cDNA for PCNA/cyclin was cloned and analyzed, it was found that the protein consisted of 261 amino acids with a calculated molecular weight of 28, 700 and the amino acid sequence was well conserved during evolution both in animal and plant kingdoms. The highly homologous nature of PCNA/cyclin suggests that the protein plays an essential role in DNA replication in eukaryotes.

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Location of fodrin in the renal tubular epithelium, the epidermal keratinocyte, and the adrenal chromaffin cell was examined by immunofluorescence and immunoelectron microscopy of frozen sections. In most of the renal epithelia, fodrin was confined to the plasma membrane and the terminal web, but in the principal cell of the collecting tubule, it was seen diffusely in the cytoplasm except for the Golgi region. In the epidermis in vivo, fodrin was observed in the cell periphery in the spinous layer, but in the cytoplasm in the basal layer. In a transformed keratinocyte cell line (PAM212), the protein was seen only in the cell surface cultured in standard Ca²⁺, but in low Ca²⁺, it was found in the cytoplasm. In the adrenal chromaffin cell in vivo, fodrin was observed only in the plasma membrane, but when massive secretion was induced by insulin, it came to be seen in newly-formed vesicles and vacuoles; a secretory granule membrane protein, cytochrome b561, was also localized in the vesicle, but not in the vacuole. The coexistence of the two proteins in the vesicle was confirmed by the positive labeling of cytochrome b561 in subcellular particles adsorbed to polyacrylamide beads coated with anti-fodrin antibody. The present study showed that fodrin is not always a plasma membrane component and that it could be distributed in the cytoplasm when cells are exposed to various stimuli.

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The ultrathin cryo-section has several advantages in the detection of enzyme activities at the light and electron microscopic levels, as it permits quick freeze fixation, and affords better penetration of the reaction medium. To establish a reliable technique for enzyme histochemistry, good visualization of ultrastructure and good retention of enzyme activities are necessary. The authors applied a covered negative staining with silicotungstate to visualize fine morphology on the ultrathin cryo-sections which demonstrated enzyme activity, after treatment with yellow ammonium sulfide. This technique makes it possible to demonstrate different enzyme activities on the same organelle by means of serial sections.
In decalcified bone tissues, alkaline phosphatase activity is detectable on ultrathin cryo-sections but not on 40μm sections without the magnesium reactivation procedure required for the usual enzyme histochemistry.

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Quick freezing followed by substitution fixation may stop all molecular movements and thus preserve all molecules in the cell as in the living condition. Therefore, it might be said that among many fixations, freeze substitution is ideal for histochemistry. In my laboratory, freeze substitution was first applied to electron microscopical enzyme histochemistry, and it proved able to preserve enough enzyme activities on an excellent ultrastructure by using glutaraldehyde and acrolein as freeze substitution fixatives.
The combination of glutaraldehyde and acrolein with substitution fixation was histochemically successful. As the pure morphology of freeze substitution fixation gains high contrast on biological membranes with osmium tetroxide, attempts were made to investigate the process and the propriety of glutaraldehyde and acrolein in freeze substitution fixation and compared with immersion fixation of conventional chemical fixation. Explorations were also made to get better contrast for the visualization of enzyme localization by electron microscopy. As the glutaraldehyde and acrolein in longer fixation time at room temperature showed enough enzyme activities, freeze substitution enzyme histochemistry could be a reliable method for enzyme histochemistry, allowing us to see the actual site of enzyme action close to the living state.

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A transient rise in the concentration of Ca²⁺ in the cortex upon ferttilization was demonstrated in echinoderm, teleost, amphlian and mammalian eggs by the luminescence of aequorin which had been microinjected into the egg, with a high sensitivity video-microscopic system. The Ca²⁺ increase started near the site of sperm entry and was propagated as a wave along the cortex toward the antipode of the egg. The wave occurred once and the propagation velocity of the wave was about 10μn/sec in echinoderm, teleost and amphibian eggs at room temperatures, while a series of Ca²⁺ waves with a higher propagation velocity were observed in golden hamster eggs in a warmed condition. Possible involvement of Ca²⁺ and inositol 1, 4, 5-triphosphate in the activation upon fertilization was investigated.

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