Light and electron microscopic immunocytochemical localization of clathrin in rat cerebellum and kidney

The precise cellular and subcellular locations of coated vesicle protein, clathrin, in rat kidney and cerebellum have been visualized by immunocytochemical techniques. In the renal tubular epithelia, clathrin-positive products were found on both free ribosomes and on those attached to rough endoplasmic reticulum (RER) and the nuclear envelope. No clathrin was observed in the cisternae of RER or the Golgi apparatus. Clathrin-positive reaction products could also be seen on coated pits, coated vesicles, Golgi-associated vesicles, basolateral cell membrane, the ground substance, and in the autophagic vacuoles. In cerebellar Purkinje and granule cell bodies, reaction products were seen localized on coated vesicles, on the budding areas from the Golgi-associated membrane and Golgi-associated vesicles. Furthermore, the membrane of the multivesicular body, the bound-ribosomes, and the ground substance were also stained. In the myelinated axon, the clathrin appeared to be concentrated on certain segments and seemed to fill in the space between neurotubules and some vesicles. In certain synaptic terminals clathrin was often seen attached to presynaptic vesicles, presynaptic membrane, and post-synaptic membrane. However, in most mossy fibers, some synaptic vesicles were not stained. These observations suggest that clathrin is synthesized on bound and free ribosomes and discharged into the cytosol where it becomes associated with a variety of ground substances and assembles on coated pits, coated vesicles, Golgi-associated vesicles, presynaptic vesicles, and pre- and postsynaptic membranes. Clathrin may be finally degraded in autophagic vacuoles.     

Light and electron microscopic studies in the cerebellum of "trembler" mutant of chickens

Light and electron microscopic analyses of the cerebellar cortex were carried out in inbred trembler mutants of Barred Plymouth Rock (BPR), crossbred trembler (CBT) and crossbred normal (CBN) chickens 6 and 34 days after hatching. The size of the cerebellum of BPR was markedly reduced, but the pattern of fissures appeared essentially normal. Both the molecular and granular layer in the BPR were much thinner than in the CBT and CBN. Ectopic Purkinje cells were observed in the molecular and granular layers, and even in the medulla. The Purkinje cells were more intensely stained in Nissl preparations than those of CBT and CBN. By 34 days, most of the Purkinje cells, which were reduced in size, contained spherical and swollen mitochondria, an undeveloped Golgi apparatus, and many stacks of rough endoplasmic reticulum (ER). The basket cells were reduced in size and exhibited a decrease in stacks of rough ER and an increase in the area of the Golgi apparatus. They contained the swollen mitochondria that were observed in the Purkinje cells. The granule cells were reduced in size, and showed a decrease in the number of cell organelles. The disorder appeared to be intrinsic to Purkinje cells since many types of degeneration were observed in the BPR Purkinje cells.     

Light and electron microscopic studies on osmoregulatory tissue in the developing brown shrimp, Penaeus aztecus

In larval and early postlarval brown shrimp, Penaeus aztecus, portions of the branchial chamber are lined by a tissue which appears ultrastructurally to be modified for osmoregulation. The distribution of this tissue, the larval stages in which it occurs, and its appearance with the light and electron microscope are presented. The significance of the distribution and ultrastructure of this modified tissue is discussed.     

Immunocytochemical localization of cathepsin H in rat kidney. Light and electron microscopic study

Light and electron microscopic localization of cathepsin H in rat kidney was studied using post-embedding immunocytochemical techniques. For light 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.     

Light and electron microscopic studies on the localization of steroid-binding protein (SBP) in rabbit spermatozoa

Light (fluorescence) and electron microscopic studies were carried out to localize steroid-binding protein (SBP) in rabbit spermatozoa. Both nonpermeabilized and permeabilized (with Tween 20, saponin, or cold acetone) spermatozoa showed fluorescence following treatment with antirabbit SBP (anti-rSBP) and subsequently with rabbit antisheep immunoglobulin G-fluorescein isothiocyanate. While the ejaculated spermatozoa were positive, epididymal sperm were observed to be negative. Although the pattern of localization of rSBP was variable, the occurrence of a negative equatorial region as well as the presence of an intense positive spherical profile ("spot") at the junction of the head and midpiece were notably consistent. The intensity of labeling with the probe, both at light and electron microscopic level, was maximal following permeabilization with cold acetone. A possible role of SBP as a steroid carrier protein across the plasma membrane of the sperm has been suggested.     

Light and electron microscopic localization of silver in biological tissue

Summary A method is described that visualizes trace amounts of silver in frozen, paraffin and epon sections from biological tissue. After exposure to light, which ensures reduction of silver ions that are not bound to sulphide, histological sections from animals treated with silver compounds are exposed to a photographic developer containing silver ions. Tissue silver acts as a catalyst for the hydroquinone reduction of silver ions to metallic silver which then accumulates at the site of the trace deposit. Light and electron micrographs showing silver in different organs from albino rats treated with silver lactate are presented. Localization of silver in motor neurons of the spinal gray matter and pons indicates a transport of silver over the blood-brain barrier. Silver precipitates in fetal liver suggest that silver ions can penetrate the placental barrier.     

Light and electron microscopic localization of atrial natriuretic peptide in the heart of spontaneously hypertensive rat

Atrial natriuretic peptide (ANP) is a newly discovered peptide hormone present mainly in the atria. We investigated the occurrence and distribution of ANP immunoreactivity in the myocardiocytes of the ventricles of spontaneously hypertensive rats by use of immunocytochemistry at both light and electron microscopic level. ANP immunoreactivity was found in the specific granules in the cytoplasm of the cardiocytes in the subendocardium and the myocardium of the ventricles, as well as in the atria. The specific granules found in the ventricles of hypertensive rats were similar in size, shape, and ANP immunoreactive content to those in the atria. The abundance of ANP immunoreactivity in the left ventricle is greater than that in the right, and appears to increase with increasing severity of hypertension. Conversely, the overall content of ANP in the atria of hypertensive rats was decreased when compared with that in age-matched normotensive rats. The present findings indicate that ventricles may become a major source for ANP synthesis and release during hypertension, and may play important roles in cardiac endocrine pathology and cardiac hypertrophy.     

Light and electron microscopic studies of postcapillary venules in developing human fetal lymph nodes.

Developing lymph nodes from 30 human fetuses with crownrump lengths (CRL) of 38 mm (8.7 wk) to 245 mm (26 wk) were studied by light and electron microscopy. Blood vessels that appear to be postcapillary venules (PCV) are present in nodes of 47 mm CRL and older fetuses. These venules first appear in nodes whehn the nodal population of lymphocytes is sparse. In these early nodes PCV are distributed randomly and consist of a low endothelium, underlying basal lamina and incomplete pericyte sheath. Early nodal PCV are distinguised from other nodal blood vessels by the presence of lymphocyte diapedesis and several luminal lymphocytes. In the late stages of nodal development PCV are the more common non-capillary blood vessel and appear in the parenchyma near the periphery of the node. Late nodal PCV are generally characterized by a cuboidal endothelium that is rich in Golgi apparatus, lysosomes and Weibel-Palade bodies. The lumen and wall of late nodal PCV contain lymphocytes. The relationship between the development of the parenchyma of fetal nodes and the appearance and activity of PCV, the passage of lymphocytes through the PCV wall and the fine structure of developing PCV are described. It is suggested that the lymphocytes that first appear in developing nodes, and the majority of the lymphocytes found in late nodes, migrate to the node via the blood vascular system and enter the nodal parenchyma by passing across PCV endothelium.     

Light and electron microscopic studies of the ventricular wall

Summary Electron microscopic data confirm the results gained with rapid Golgi preparations of adult rodent brains that tanycytes occur in clusters along the lateral wall of the third ventricle. The cytoplasmic matrix of these cells is considerably denser than that of typical ependymal cells. They have filaments and microtubules throughout their cytoplasm along with mitochondria and polysomes. At the surface is a compact group of microvilli which suggest that tanycytes might selectively absorb material from the ventricle.The tanycytes are segregated from neuropil by other tanycyte processes, by neighboring ependymal cells and by astrocytes. Yet there are gaps in this sheath. At these points tanycytes either abut upon or surround nonglial components of the neural fabric.Their cytological features and relations with the neuropil suggest that tanycytes selectively absorb material from the ventricle and release it along the basal process, primarily affecting those segments of neurons immediately adjacent to the tanycyte.Supported by: NINDS Grants 5 R01 NS 09001-02 NEUA, 5T01 NB 5309, and GM 00958, and by the Eleanor Roosevelt Cancer Foundation Research Institute.Acknowledgements: This work was initiated in the Anatomy Department of the Harvard Medical School with facilities provided by Prof. S. L. Palay (U.S. Public Health Service Grant No. NB 05591). Dr. R. B. Wuerker kindly and patiently provided the instruction and orientation to electron microscopy. The major portion of the study was completed in the Neurology Department of the University of Utah with the extremely competent, challenging assistance of Dee Lerdahl, Nina Belgarian, Keith Johnson and Lynn Kendricks.     

Light and electron microscopic studies on experimental nocardia-toxicosis in mice

An exotoxin (HS-6) produced by Nocardia otitidiscaviarum isolated from certain lesions of cutaneous nocardiosis of a male 82-year-old patient induced severe injuries in the pancreas, liver, stomach, small intestine, heart, thymus and kidney of male ICR mice. Mice given Nocardia-free preparation of HS-6 at a dose of 1 mg/kg of body weight developed several autophagic vacuoles in the pancreas and liver within 20 min after the i.p. injection. Thereafter, the autophagic vacuoles increased in number and size with time. About 24 hr after the administration of HS-6, the liver showed marked accumulation of fat droplets in the cytoplasm of the hepatocytes. Although they contained abundant autophagic vacuoles in the regions of RER, there were no lipomatoses in the acinar cells of the pancreas, those of the chief cells and smooth muscle cells of the stomach, Paneth cells, goblet cells, smooth muscle cells of the small intestine, and plasma cells in the digestive tract. Biochemical examinations revealed that HS-6 had no significant effect on the protein synthesis of reticulocytes. Inoculation of the Nocardia into the mouse peritoneal cavities caused marked granulomatoses in the pancreas, liver and regional lymph nodes, but did not develop autophagic vacuoles in RER regions of these organs.     

Light and electron microscopic localization of L-alpha-hydroxyacid oxidase in rat kidney revealed by immunocytochemical techniques

Light and electron microscopic localization of L-alpha-hydroxyacid oxidase (L-HOX) in rat kidney was studied by means of immunocytochemical techniques. Isozymes A and B of L-HOX were purified from rat liver and kidney, respectively. The apparent molecular weights of the subunits of the isozymes A and B were 35,800 and 33,500 daltons, respectively, by a slab gel electrophoresis. Antibodies to the isozymes were raised in rabbits. Anti(isozyme A) is not cross-reactive with the isozyme B and vice versa anti(isozyme B) not with the isozyme A. Using anti-isozyme B, semithin sections of Epon-embedded material and ultrathin sections of Lowicryl K4M-embedded material were stained by immunoenzyme and protein A-gold techniques, respectively. By light microscopy, fine discrete granular staining was noted in proximal tubules, but not in distal tubules including thick and thin limbs of Henle and collecting tubules. By electron microscopy, gold particles representing the antigen sites for L-HOX B were confined exclusively to peroxisomes, in which most of the gold particles were localized in electron dense peripheral matrix, but little in central matrix with low electron density. The results indicate that L-HOX B does not homogeneously distribute in peroxisomes of rat kidney but might be associated with some substructure within peroxisome matrix.     

Immunocytochemical localization of ornithine transcarbamylase in rat intestinal mucosa. Light and electron microscopic study

We investigated light and electron microscopic localization of ornithine transcarbamylase (OTC) in rat intestinal mucosa. In the immunoblotting assay of OTC-related protein, a single protein band with a molecular weight of about 36,500 is observed in extracts of liver and small intestinal mucosa but is not observed in those of stomach and large intestine. For light microscopy, tissue slices of the digestive system were embedded in Epon and stained by using anti-bovine OTC rabbit IgG and the immunoenzyme technique. For electron microscopy, slices of these and the liver tissues were embedded in Lowicryl K4M and stained by the protein A-gold technique. By light microscopy, the absorptive epithelial cells of duodenum, jejunum, and ileum stained positively for OTC, but stomach, large intestine, rectum, and propria mucosa of small intestine were not stained. Electron microscopy showed that gold particles representing the antigenic sites for OTC were confined to the mitochondrial matrix of hepatocytes and small intestinal epithelial cells. However, the enzyme was detected in mitochondria of neither liver endothelial cells, submucosal cells of small intestine, nor large intestinal epithelial cells. Labeling density of mitochondria in the absorptive epithelial cells of duodenum, jejunum, and ileum was about half of that in liver cells.     

Light and electron microscopic localization of l-alpha-hydroxyacid oxidase in rat kidney revealed by immunocytochemical techniques

Summary Light and electron microscopic localization of l-alpha-hydroxyacid oxidase (l-HOX) in rat kidney was studied by means of immunocytochemical · techniques. Isozymes A and B of l-HOX were purified from rat liver and kidney, respectively. The apparent molecular weights of the subunits of the isozymes A and B were 35,800 and 33,500 daltons, respectively, by a slab gel electrophoresis. Antibodies to the isozymes were raised in rabbits. Anti(isozyme A) is not cross-reactive with the isozyme B and vice versa anti(isozyme B) not with the isozyme A. Using anti-isozyme B, semithin sections of Epon-embedded material and ultrathin sections of Lowicryl K4M-embedded material were stained by immunoenzyme and protein A-gold techniques, respectively. By light microscopy, fine discrete granular staining was noted in proximal tubules, but not in distal tubules including thick and thin limbs of Henle and collecting tubules. By electron microscopy, gold particles representing the antigen sites for l-HOX B were confined exclusively to peroxisomes, in which most of the gold particles were localized in electron dense peripheral matrix, but little in central matrix with low electron density. The results indicate that l-HOX B does not homogeneously distribute in peroxisomes of rat kidney but might be associated with some substructure within peroxisome matrix.     

Light and electron microscopic studies of crayfish hemocytes

Using light and electron microscopy, three hemocyte types are described in the hemolymph of the crayfish. The coagulocyte comprises 65% of the total hemocyte number and contains medium-sized cytoplasmic granules, abundant dilated rough endoplasmic reticulum, and a highly developed Golgi complex. It rapidly undergoes cytolysis in vitro and participates in coagulation by releasing the contents of its granules to the hemolymph. The granulocyte comprises 31% of the total hemocyte number and is capable of phagocytosis. It contains large, irregularly shaped cytoplasmic granules, a moderately developed Golgi complex, and moderate amounts of non-dilated rough endoplasmic reticulum. During coagulation in vitro, the cell attaches and spreads onto the substratum; this is followed by a slow intracellular granule breakdown and cytolysis. The amebocyte comprises 4% of the total hemocyte number and it is also capable of phagocytosis. It possesses small cytoplasmic granules, many vacuoles, a moderately developed Golgi complex, and large amounts of smooth endoplasmic reticulum. It is distinguished from the other two cell types by being stable and motile in vitro.