Immunocytochemical localization of rabbit gastric lipase and pepsinogen

Lipase and pepsin activities were determined in rabbit gastric biopsy specimens. Lipase activity was found to be restricted to a small part of the fundic mucosa, near the cardia, whereas pepsin activity spread over about two thirds of the total fundic area, overlapping that of lipase. The cells producing these two enzymes were labeled by immunofluorescence using polyclonal antibodies against rabbit gastric lipase (RGL) or antibodies against rabbit pepsinogen. The immunocytochemical localization showed unequivocally that RGL and pepsinogen, which were both present in the cardial area, were in fact located in different gastric cells. The cells producing pepsinogen were in the lower base of the gastric fundic glands, whereas the cells producing RGL were in the upper base of the same glands. The cells producing pepsinogen and RGL showed no significant morphological differences. In the part of the fundic area, where only pepsin activity was detected, cells producing pepsinogen covered both the lower and the upper base of the gastric glands. No chief cells were observed in the antral mucosa. RGL and pepsinogen could represent useful gastric enzyme markers for cellular differentiation studies.     

Immunocytochemical localization of cyclooxygenase-1 and cyclooxygenase-2 in the rat stomach

Summary Prostaglandins are considered to play important roles in gastric mucosal protection. The rate-limiting enzyme involved in the biosynthesis of prostaglandins is cyclooxygenase (COX), also known as prostaglandin H synthase. Two forms of COX are known: a constitutively expressed form (COX-1) and a newly-characterized, inducible form (COX-2). In the present study, the immunocytochemical localization of COX-1 and COX-2 was examined in the rat gastrointestinal tract. A strong immunoreactivity for COX-1 was localized in the mucous neck cells of gastric gland. A weak reactivity for COX-1 was also found in the mucous cell types in the cardiac gland and pyloric gland of the stomach as well as in the Brunner's gland of duodenum. Ultrastructurally, the immunoreactivity was localized to the apical cytoplasm of these cells. On the other hand, immunoreactivity for COX-2 was distributed in the surface mucous cells in both the fundic and pyloric regions of stomach. These results suggest that a subset of mucous cells is the primary site for production of prostaglandins in the rat gastrointestinal tract, and that two forms of COX are expressed in distinct types of mucous cell.     

Immunocytochemical localization of aromatase in rat testis

Summary The immunocytochemical localization of armatase in the testes of young and adult rats was investigated by an indirect-immunofluorescent method using antihuman placental aromatase-II cytochrome P-450 antibody. In both young (1 and 2 weeks old) and adult rats, only the Leydig cells in the interstitial tissue showed a positive immunoreaction for aromatase, while the germ cells and Sertoli cells in the seminiferous tubule were entirely negative. In addition, electron microscopy revealed that the Leydig cells in the testes of young as well as adult rats have a well-developed smooth endoplasmic reticulum, mitochondria with tubulovesicular cristae, and a few lipid droplets; these structures being characteristic of steroid secretory cells. On the basis of these results, we suggest that estrogens are mainly synthesized in Leydig cells of the testes.Supported by grants from the Ministry of Education, Science, and Culture, Japan, and USPHS HD 04945     

Immunocytochemical localization of aromatase in rat testis

The immunocytochemical localization of aromatase in the testes of young and adult rats was investigated by an indirect-immunofluorescent method using antihuman placental aromatase-II cytochrome P-450 antibody. In both young (1 and 2 weeks old) and adult rats, only the Leydig cells in the interstitial tissue showed a positive immunoreaction for aromatase, while the germ cells and Sertoli cells in the seminiferous tubule were entirely negative. In addition, electron microscopy revealed that the Leydig cells in the testes of young as well as adult rats have a well-developed smooth endoplasmic reticulum, mitochondria with tubulovesicular cristae, and a few lipid droplets, these structures being characteristic of steroid secretory cells. On the basis of these results, we suggest that estrogens are mainly synthesized in Leydig cells of the testes.     

Immunocytochemical localization of glucocorticoid receptor in rat skin

The aim of the present study was to map immunohistochemically the distribution of the glucocorticoid receptor (GR) in rat skin. Nuclear GR-like immunoreactivity (LI) was found in both epidermis and dermis. In the epidermis, the basal cell layer showed an intense immunoreaction; the lower part of the spinous layer was also labelled. In the dermis, the fibroblasts as well as the sweat glands, sebocytes and adipocytes were GR-immunoreactive (IR). In the root sheath of the hair follicle the staining was most intensive in the outer layer. The endothelial cells comprising the smooth muscle cells of the blood vessels, as well as the arrector pili muscle, showed GR-LI. In the peripheral nerves, the immunoreaction was localized to the nuclei of the Schwann cells and in the perineurial fibroblasts. Mast cells did not show nuclear GR-LI. Based on our immunocytochemical findings that several cell types of the skin are GR-IR, the variable physiological and pharmacological effects of glucocorticoids are easier to understand.     

Immunocytochemical localization of acid phosphatase in rat liver

Localization of acid phosphatase (ACPase) in rat liver was investigated by immunocytochemical techniques. Rat liver was fixed by perfusion and cut into thick tissue slices, which were embedded in Epon or Lowicryl K4M. For light microscopy (LM), semithin Epon sections were stained for the enzyme ACPase by an indirect immunoenzyme technique. For electron microscopy (EM), ultra-thin Lowicryl K4M sections were stained by a protein A-gold technique. By means of LM, granular reaction deposits were observed in hepatocytes and sinus-lining cells. Stained granules were present in the juxtanuclear cytoplasm, but they did not correspond to a typical staining pattern for the Golgi complex. EM revealed that gold particles indicating ACPase antigens were present on lysosomes and on some vesicles locating in the trans Golgi region. Endosomelike vesicles were strongly positive for the labeling. Golgi cisterna were mostly negative, but weak signals were noted in dilated sacules. The plasma membranes on the sinusoidal and bile canalicular sides were labeled by a few gold particles. The results indicate that ACPase is present in endosomes and in a restricted area of plasma membrane, as well as in the lysosomal system.     

Immunocytochemical localization of lysosomal beta-galactosidase in rat liver

beta-galactosidase is a ubiquitous lysosomal hydrolase that specifically cleaves terminal beta-galactosyl residues from glycoproteins, glycosaminoglycans, oligosaccharides, and glycolipids. To study the intracellular distribution of this enzyme, we prepared a specific polyclonal antibody to lysosomal beta-galactosidase by immunizing rabbits with a highly purified preparation of beta- galactosidase from rat liver. Using this antibody we employed an immunocytochemical technique (protein A coupled to horseradish peroxidase and diaminobenzidine cytochemistry) and showed that beta- galactosidase is present in all hepatocytes of the rat liver. All types of lysosomes, the rough endoplasmic reticulum, and the specialized region of smooth endoplasmic reticulum known as GERL showed immunoreactivity. This in situ distribution suggests that these organelles are involved in the biosynthesis and intracellular sorting of this lysosomal enzyme.     

Immunocytochemical localization of cathepsin H in rat kidney

Summary Light and electron microscopic localization of cathepsin H in rat kidney was studied using post-embedding immunocytochemical techniques. For ligh microscopy, Epon sections of the kidney were stained by immunoenzyme method after removal of Epon and for electron microscopy, ultrathin sections of the Lowicryl K4M-embedded material were labeled by protein A-gold (pAg) technique. By light microscopy, fine granular staining was found in throughout the nephron, but the staining intensity considerably varied. The strongest staining was noted in the S1 segment of the proximal tubules followed by the S2 and S3 segments and the medullary collecting tubules. The glomeruli, the distal tubules, and the cortical collecting tubules were weakly stained. By electron microscopy, a gold label was found exclusively in lysosomes, which showed various sizes and labeling intensity. The results were quite consistent with the light microscopic results. The labeling intensity tended to increase as the matrix of lysosomes was condensed. Quantitative analysis of the labeling density of lysosomes demonstrated that the highest labeling density is found in the S1 segment of the proximal tubules and the labeling density of other renal segments is significantly low levels. The results indicate that a main site for cathepsin H in rat kidney is the S1 segment of the proximal tubules.     

Immunocytochemical study of pepsinogen 1-producing cells in the fundic mucosa of the stomach in developing mice

Summary Development and maturation of pepsinogen 1-producing cells were studied in the gastric fundic mucosa of the mouse by means of light- and electron-microscopic immunocytochemistry using rabbit anti-rat pepsinogen 1-serum. In the adult mouse, secretory granules in mucous neck cells, transitional mucous neck/chief cells and chief cells are immunolabeled. The numerical density of gold particles on zymogen granules is not significantly altered among different stages of maturation of chief cells. In addition, rough endoplasmic reticulum and Golgi complex of these cell types show a weak labeling. In mice from day 16 of gestation to postnatal day 14 mucous neck cells and chief cells cannot be distinguished, but only one type of pepsinogen 1-producing cell, called primitive chief cell, is identified in the fundic gland. The intensity of immunoreactivity of secretory granules in primitive chief cells is uniform within an individual cell but varies greatly among different cells. The majority of primitive chief cells contains weakly labeled granules regardless of the maturation stage of cells or of animals. On postnatal day 21, mucous neck, transitional and chief cells are distinguishable, and secretory granules in these cells are intensely immunolabeled as in the adult. These results suggest that pepsinogen 1-production rapidly increases with differentiation of mucouse neck and chief cells.     

Immunocytochemical localization of cathepsin D in the rat osteoclast

Summary We performed immunocytochemical localization of cathepsin D in osteoclasts of the proximal growth plate of the rat femurs using both the avidin-biotin-peroxidase complex method for cryo-semi-thin (1 m) sections and the colloidal gold-labeled IgG method for K4M ultra-thin sections.At the light microscopic level, cathepsin D immunoreactivity in the osteoclasts appeared at the vesicles, granules, and/or small vacuoles. They were distributed throughout the cytoplasm of each cell and were relatively numerous close to the bone surface. This antigen could not be detected at the eroded bone surface. As for other cells, immunoreactivity was seen only in the lysosomes of osteoblast-like cells. Immunoreactivity in the osteoclasts was stronger and greater in the density and number than in osteoblast-like cells. At the electron microscopic level, osteoclasts with well-developed ruffled border possessed numerous cathepsin D-containing lysosomes, vacuoles, and coated vesicle-like structures. Cathepsin D-containing lysosomes fused with cathepsinnegative vacuoles and formed large secondary lysosomes. Osteoclasts with poorly developed ruffled border possessed fewer cathepsin D-containing lysosomes than those with well-developed ruffled border. No immunogold particles were seen in vacuole-like channel expansions of the ruffled borders, between the channels of the ruffled borders, or on the eroded bone surface.These findings demonstrate that osteoclasts contain a large amount of cathepsin D. They suggest that cathepsin D is necessary for osteoclastic bone resorption, that it plays an indirect rather than direct role.     

Immunocytochemical localization of somatostatin-containing neurons in the rat hypothalamus

Summary The rat hypothalamus was studied at the light microscopic level with the use of single and double immunocytochemical staining methods. It was shown that the rat supraoptic and paraventricular hypothalamic nuclei, and their accessory neurosecretory nuclei, do not contain magnocellular somatostatin neurons. The distribution of the hypothalamic parvocellular somatostatin cells is described. The parvocellular component of the rat hypothalamic paraventricular nucleus is, at least partly, composed of somatostatin cells: they form a fairly well circumscribed periventricular cell mass. The rat suprachiasmatic nuclei contain separate somatostatin neurons and vasopressin neurons. Scattered somatostatin cells are present in the entire arcuate nucleus. In addition to the periventricular somatostatin cells located in the preopticanterior hypothalamic area and in the arcuate nucleus, the rat hypothalamus also contains numerous scattered somatostatin cells located distant from the third ventricle.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Immunocytochemical localization of d-amino acid oxidase in rat brain

d-amino acid oxidase (d-AAO) is a peroxisomal flavoenzyme, the physiological substrate and the precise function of which are still unclear. We have investigated D-AAO distribution in rat brain, by immunocytochemistry, with an affinity-purified polyclonal antibody. Immunoreactivity occurred in both neuronal and glial cells, albeit at different densities. Glial immunostaning was strongest in the caudal brainstem and cerebellar cortex, particularly in astrocytes, Golgi-Bergmann glia, and tanycytes. Hindbrain neurons were generally more immunoreactive than those in the forebrain. Immunopositive forebrain cell populations included mitral cells in the olfactory bulb, cortical and hippocampal neurons, ventral pallidum, and septal, reticular thalamic, and paraventricular hypothalamic nuclei. Within the positive regions, not all the neuronal populations were equally immunoreactive; for example, in the thalamus, only the reticular and anterodorsal nuclei showed intense labelling. In the hindbrain, immunopositivity was virtually ubiquitous, and was especially strong in the reticular formation, pontine, ventral and dorsal cochlear, vestibular, cranial motor nuclei, deep cerebellar nuclei, and the cerebellar cortex, especially in Golgi and Purkinje cells.     

Immunocytochemical studies on the localization of pancreatic-type phospholipase A2 in rat stomach and pancreas, with special reference to the stomach cells

Using a specific polyclonal antibody raised against rat pancreatic phospholipase A2 (PLA2), we investigated the localization of the enzyme in the rat pancreas and stomach by light and electron microscopy. In the pancreas, the enzyme was localized in the acinar cells, whereas the pancreatic islets showed no immunoreaction. In the stomach, the PLA2 reactive with the anti-pancreatic PLA2 antibody was distributed exclusively in the gastric glands, but not in the gastric pits or the pyloric glands. On the section of the stomach subjected to immuno- and PAS-staining, immunopositive cells were not the PAS-positive cells located in the gastric pit and the neck region of the gastric gland. Immunopositive cells were present from the neck to the bottom of the gastric gland. Immunoelectron microscopic observation revealed that the immunogold-labeled cell had a highly-developed rough endoplasmic reticulum in the basal cytoplasm and characteristic zymogen granules in the apical cytoplasm. Taking into account the cell position in the gastric gland, the immunopositive cell could therefore be identified as a chief cell. Since no double stainability with PLA2 and PAS was observed in the same cell, it is suggested that PLA2 could be used cytochemically as a marker enzyme of the chief cell in the gastric gland at the light-microscopic level. From the immunoelectron microscopic findings, we believe that the PLA2 in the stomach is released into the lumen of the stomach by exocytosis and could function as a digestive enzyme in the alimentary tract, like the PLA2 secreted from the pancreas. Other possible roles of the PLA2 in the stomach are discussed.     

Immunocytochemical localization of tissuebound oestradiol in rat paracervical ganglion

Summary The uterine paracervical ganglion (Frankenhauser's ganglion) contains the terminal neurons of the cholinergic sacral parasympathetic, the short adrenergic sympathetic and the peptideric (vasoactive intestinal polypeptide-containing) nerves of the internal genitalia. Previous studies have shown that either the number of cells or transmitter content of each of these neuronal systems is altered by variations in steroid hormones. Furthermore, our recent study showed that some component of the rat paracervical ganglion was capable of metabolizing [³H]oestradiol to oestrone and the 2-OH and 4-OH forms of oestrone and oestradiol. The present study employs the peroxidase-anti-peroxidase immunohistochemical method to localize oestradiol in rat paracervical ganglia. Specific reaction product was identified in (1) cytoplasm and some nuclei of principal ganglion cells, (2) cytoplasm of large vacuolated ganglion cells, (3) cytoplasm of'small intensely fluorescent' cells and (4) some nerve fibres in ganglia from animals in oestrus. The cytoplasm of principal neurons and some nerve fibres exhibited specific staining for oestradiol in dioestrus and pro-oestrus. No oestradiol was localized in ganglia excised from animals in metoestrus. Preincubation in oestradiol before fixation was necessary for specific localization of oestradiol; treatment of tissues with oestradiol after fixation was not required. These results are not consistent with binding of oestradiol to the classical oestrogen receptor. The resistance of oestradiol to organic solvent extraction suggests that oestradiol is covalently bound to tissue proteins. Such covalently bound oestradiol has been reported as a by-product of tissue metabolism of oestradiol via P-450 enzymes.     

Immunocytochemical localization of angiotensinogen in rat liver and kidney

Cell and Tissue Research - The renin substrate, angiotensinogen, was localized by immunocytochemistry in liver and kidney of normal rats by the use of an antiserum directed against pure rat...     

Immunocytochemical studies on the localization of pancreatic-type phospholipase A2 in rat stomach and pancreas,with special reference to the stomach cells

Summary Using a specific polyclonal antibody raised against rat pancreatic phospholipase A₂ (PLA₂), we investigated the localization of the enzyme in the rat pancreas and stomach by light and electron microscopy. In the pancreas, the enzyme was localized in the acinar cells, whereas the pancreatic islets showed no immunoreaction. In the stomach, the PLA₂ reactive with the anti-pancreatic PLA₂ antibody was distributed exclusively in the gastric glands, but not in the gastric pits or the pyloric glands. On the section of the stomach subjected to immuno- and PAS-staining, immunopositive cells were not the PAS-positive cells located in the gastric pit and the neck region of the gastric gland. Immunopositive cells were present from the neck to the bottom of the gastric gland. Immunoelectron microscopic observation revealed that the immunogold-labeled cell had a highly-developed rough endoplasmic reticulum in the basal cytoplasm and characteristic zymogen granules in the apical cytoplasm. Taking into account the cell position in the gastric gland, the immunopositive cell could therefore be identified as a chief cell. Since no double stainability with PLA₂ and PAS was observed in the same cell, it is suggested that PLA₂ could be used cytochemically as a marker enzyme of the chief cell in the gastric gland at the light-microscopic level. From the immunoelectron microscopic findings, we believe that the PLA₂ in the stomach is released into the lumen of the stomach by exocytosis and could function as a digestive enzyme in the alimentary tract, like the PLA₂ secreted from the pancreas. Other possible roles of the PLA₂ in the stomach are discussed.     

Immunocytochemical and enzyme histochemical localization of kallikrein-like enzymes in colon, intestine, and stomach of rat and cat

Kallikrein was localized in goblet (or mucous) cells of rat colon and in rat and cat small intestine and stomach by two immunocytochemical techniques. A kallikrein-like enzyme was also localized by enzyme histochemistry in mast cells of colon, intestine, and stomach of the cat, where they appeared to be associated with blood vessels in the lamina propria. The mast cell enzyme, however, was not detected by immunocytochemistry using antibodies to kallikrein. Modification in the enzyme histochemical procedure (pH, fixation) yielded positive results for a kallikrein-like protease in goblet cells of the intestine and colon. The possible physiological and pathological significance of kallikrein-like enzyme in the gastrointestinal tract and elsewhere is discussed.     

Immunocytochemical localization of actin in the nucleolus of rat oocytes

Summary— In order to determine the localization of actin, growing and fully grown rat oocytes were immunocytochemically examined using a post-embedding ultrastructural protein-A gold technique. In quiescent oocytes, the nucleoplasm showed slightly lower levels of actin signal when compared to the surrounding cytoplasm. The highest levels of labeling were found on nucleoli showing a reticular type morphology. In oocytes at the diakinesis stage in which nucleolar compaction had occurred, the levels of labeling increased by 5–6 times those found in quiescent oocytes. Except for conspicuous accumulation of actin under the plasma membrane, compact nucleoli had significantly higher levels of labeling when compared with those found on the general cytoplasm, while the nucleoplasm with homogeneously dispersed chromatin showed significantly lower levels of associated actin signal than the general cytoplasm. In oocytes at metaphase I, the cytoplasmic region had comparable or lower levels of labeling than the cytoplasm of oocytes at diakinesis. The meiotic spindle embedded in material with medium electron density showed a similar level of labeling as the surrounding cytoplasm. On the other hand, significantly higher levels of associated actin were observed on the chromosomes of metaphase I. The actin signals were dispersed over the chromosomes and not concentrated on a specific region. These results suggest that nuclear actin may be involved in the process of chromosome construction and also the formation of the compacted structure of the nucleolus.     

Immunocytochemical localization of exocrine enzymes in rat pancreatic acinar carcinoma

Ten pancreatic secretory proteins have been demonstrated in differentiated pancreatic acinar carcinoma cells by the protein A-gold immunocytochemical approach. The high resolution of the technique has allowed for the localization of the different proteins in the cellular compartments involved in protein secretion: RER, Golgi and secretory granules. The quantitative evaluation of the labeling for amylase has demonstrated the presence of an increasing gradient in the intensity from the RER to the Golgi and to the secretory granules which may reflect the process of protein concentration along the secretory pathway. These results, together with those obtained using the pulse-labeling autoradiographic approach, demonstrate that differentiated acinar carcinoma cells are capable of processing secretory proteins. When intensities of labeling obtained for different proteins on acinar carcinoma cells were compared to those obtained on normal pancreatic acinar cells, major differences were observed for some proteins. In addition, studies performed on the pancreatic tissue of the tumor-bearing animals have shown the presence of morphological alterations in the acinar cells.     

Immunocytochemical localization of liver-specific proteins in pancreatic hepatocytes of rat

Hepatocytes are induced in the pancreas of rats maintained first on a copper-deficient diet for 8 weeks and then on normal rat chow. These cells are morphologically identical to parenchymal cells of the liver. These hepatocytes contain two liver-specific proteins: carbamyl phosphate synthetase I, a mitochondrial matrix protein that participates in the conversion of ammonia to carbamyl phosphate; and urate oxidase, an enzyme that catalyzes the oxidation of uric acid to allantoin. In addition, we also present evidence indicating that dietary administration of ciprofibrate induces peroxisomal beta-oxidation pathway enzymes, while the levels of catalase are unaltered in pancreatic hepatocytes. These observations along with the previously published results further establish the identity of pancreatic hepatocytes to parenchymal cells of liver and clearly indicate that transdifferentiation of pancreatic cells to hepatocytes is associated with activation of several liver-specific genes.