An improved timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues

Summary Modifications of the Timm sulphide silver method for the demonstration of heavy metals are described.To improve the structural preservation of the tissues perfusion with a glutaraldehyde fixative is employed before perfusion with the sodium sulphide solution. For the subsequent staining for light and electron microscopy, procedures for plastic embedding, paraffin embedding and cryostat sectioning are presented. Examples from several tissues are shown, including the pituitary, pancreas, intestine, tongue, kidney, testis and brain. The staining of autolytic, postmortal human brain tissue is demonstrated.     

An improved Timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues.

Modifications of the Timm sulphide silver method for the demonstration of heavy metals are described. To improve the structural preservation of the tissues perfusion with a glutaraldehyde fixature is employed before perfusion with the sodium sulphide solution. For the subsequent staining for light and electron microscopy, procedures for plastic embedding, paraffin embedding and cryostat sectioning are presented. Examples from several tissues are shown, including the pituitary, pancreas, intestine, tongue, kidney, testis and brain. The staining of autolytic, postmortal human brain tissue is demonstrated.     

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 localization of GABA-like immunoreactivity in myenteric plexus of chicken

Summary Whole-mounts of 1-day-old chicken midgut were incubated with an antiserum against GABA-glutaraldehyde-BSA conjugate. The immunoreaction was visualized by using the peroxidase-antiperoxidase method, and processed for consecutive light and electronmicroscopic observation. GABA was selectively localized in some of the varicose and nonvaricose nerve fibres of the myenteric plexus. The varicose fibres formed dense networks within the myenteric ganglia, some of which — mainly in duodenum — also contained immunopositive nerve cell bodies. Some of the varicose fibres projected out from the myenteric plexus into the circular muscle layer. At the electronmicroscopic level, labelled axon terminals formed synaptic contact with unlabelled perikarya and vica versa. At the same time, no labelled terminals were found on immunostained cells. In a few cases, axon terminals with GABA positivity were situated close to the smooth muscle cells in the circular muscle layer, suggesting a prejunctional GABA effect on the neighbouring nerve terminals on the release of their transmitters.     

Light and electron microscopic localization of GABA-like immunoreactivity in myenteric plexus of chicken

Whole-mounts of 1-day-old chicken midgut were incubated with an antiserum against GABA-glutaraldehyde-BSA conjugate. The immunoreaction was visualized by using the peroxidase-antiperoxidase method, and processed for consecutive light and electronmicroscopic observation. GABA was selectively localized in some of the varicose and nonvaricose nerve fibres of the myenteric plexus. The varicose fibres formed dense networks within the myenteric ganglia, some of which--mainly in duodenum--also contained immunopositive nerve cell bodies. Some of the varicose fibres projected out from the myenteric plexus into the circular muscle layer. At the electronmicroscopic level, labelled axon terminals formed synaptic contact with unlabelled perikarya and vica versa. At the same time, no labelled terminals were found on immunostained cells. In a few cases, axon terminals with GABA positivity were situated close to the smooth muscle cells in the circular muscle layer, suggesting a prejunctional GABA effect on the neighbouring nerve terminals on the release of their transmitters.     

Light and electron microscopic immunocytochemical localization of two major proteins in garlic bulb

Garlic is known as a potent spice and a medicine with broad therapeutic properties ranging from antibacterial to anticancer, antidiabetic, and anticoagulant. Two major proteins of 40 KD and 14 KD constituting approximately 96% of total garlic proteins have been recently purified at our Institute. This immunocytochemical and ultrastructural study revealed that the 40 KD protein was localized in the parenchyma sheath cells (PSC) of garlic bulbs, whereas the 14 KD protein was present in the cortical cells (CC). Immunogold electron microscopy study indicated that the 40 KD protein was specifically localized in the globular granules of the cytoplasmic area of PSC. Each globular granule was amorphous and homogenous with membrane limiting its outermost layer. The yellowish color of PSC in freshly cut slices of garlic bulb suggested that PSC may have sulfur-containing compounds such as allicin, the primary contributor of the pungency and medicinal properties of garlic. Ellman's reagent test quantitatively revealed that there were 17.8 n moles sulfhydryl (SH)/ml of 40 KD garlic protein. Microtubule tubulin in mitotic figures from PHA-stimulated human short-term whole blood cultures reacted strongly with antitubulin antibody but reacted negatively with anti-40 KD garlic protein antibodies and therefore was not related to the 40 KD garlic protein immunocytochemically.     

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 microscopic localization of cytochemical reactions in epoxy-embedded material for electron microscopy.

Tissues from mice were fixed in 1.5% glutaraldehyde, treated for the ultrastructural localization of alkaline phosphatase or Mg++-dependent adenosine triphosphatase, post-fixed in osmium tetroxide, dehydrated and embedded in plastic for electron microscopy. The sites of reaction were visualized in 1-mu plastic sections counterstained with toluidine blue, using a phase contrast microscope. The data show a close correlation between the sites of reaction observed with the phase contrast microscope and the sites studied with the electron microscope. The use of this technique for the study of these phosphatases in normal and pathologic tissues is recommended in order to achieve a high degree of accuracy in selecting a portion of the tissue sample for electron microscopy and to obtain greater resolution in the localization of these enzymes with the light microscope.     

Transmitting tissue in the pistil of tobacco: Light and electron microscopic observations

Summary The pistil of tobacco (Nicotiana tabacum L. cv. Wisconsin 38) is comprised of two fused carpels. The stigma is bilobed, papillose, and at maturity is covered with a sticky exudate. The style is solid. Both stigma and style are made up of four tissue elements—epidermis, cortex, vascular, and transmitting tissue. Transmitting tissue in this species is chlorophyllous. Transmitting cells have thin primary walls and are separated by massive deposits of denselystaining amorphous material. The cells contain numerous mitochondria, dictyosomes, RER, amyloplasts, ribosomes, as well as crystal-containing microbodies and myelin-like formations. Observations are discussed in relation to other reports dealing with similar cell populations.Abbreviations A amyloplast - CO cortex - CCB crystal-containing bodies - D dictyosome - IS intercellular space - IM intercellular matrix - M mifochondrion - MY myelin formation - N nucleus - O ovary - P potysomes - PW primary wall - S starch grain - ST stigma - SV style - TT transmitting tissue - V vacuole - VB vascular bundle     

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 on the localization of hyaluronic acid in developing rat cerebellum

The hyaluronic acid-binding region was prepared by trypsin digestion of chondroitin sulfate proteoglycan aggregate from the Swarm rat chondrosarcoma, and biotinylated in the presence of hyaluronic acid and link protein. After isolation by gel filtration and HPLC in 4 M guanidine HCl, the biotinylated hyaluronic acid-binding region was used, in conjunction with avidin-peroxidase, as a specific probe for the light and electron microscopic localization of hyaluronic acid in developing and mature rat cerebellum. At 1 w postnatal, there is strong staining of extracellular hyaluronic acid in the presumptive white matter, in the internal granule cell layer, and as a dense band at the base of the molecular layer, surrounding the parallel fibers. This staining moves progressively towards the pial surface during the second postnatal week, and extracellular staining remains predominant through postnatal week three. In adult brain, there is no significant extracellular staining of hyaluronic acid, which is most apparent in the granule cell cytoplasm, and intra-axonally in parallel fibers and some myelinated axons. The white matter is also unstained in adult brain, and no staining was seen in Purkinje cell bodies or dendrites at any age. The localization of hyaluronic acid and its developmental changes are very similar to that previously found in immunocytochemical studies of the chondroitin sulfate proteoglycan in nervous tissue (Aquino, D. A., R. U. Margolis, and R. K. Margolis. 1984. J. Cell Biol. 99:1117-1129; Aquino, D. A., R. U. Margolis, and R. K. Margolis. J. Cell Biol. 99:1130-1139), and to recent results from studies using monoclonal antibodies to the hyaluronic acid-binding region and link protein. The presence of brain hyaluronic acid in the form of aggregates with chondroitin sulfate proteoglycans would be consistent with their similar localizations and coordinate developmental changes.     

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 ACTH and pro-opiomelanocortin-derived peptides in human developmental and neoplastic cells

Oncofetal aspects of ACTH and pro-opiomelanocortin (POMC)-derived peptides were studied immunohistochemically at the light and electron microscopic level in human fetal pituitary glands, pituitary adenomas, and small-cell carcinoma of the lung. ACTH, beta-endorphin, and gamma-MSH were localized in the same cells of both fetal and adult pituitary, as well as in the above-mentioned neoplastic tissues. However, alpha-MSH was observed only in the early fetal pituitary, its concentration decreasing with advancing gestational age. The adult pituitary contained only a few alpha-MSH-positive cells. By immunoelectron microscopy, ACTH in the adult pituitary was localized exclusively in the secretory granules. In fetal pituitary at 9 weeks' gestation, ACTH was localized in the perinuclear spaces (PNS), cisternae of rough endoplasmic reticulum (RER), Golgi saccules, and secretory granules. The staining pattern of ACTH in these organelles varied from cell to cell. In fetal pituitaries of greater gestational ages, ACTH was localized in secretory granules. The pituitary adenomas mimicked the staining characteristics of the adult pituitary, i.e., negative or only very occasional alpha-MSH staining and localization of ACTH in the secretory granules. The ectopic ACTH-producing tumors showed a staining pattern similar to that of the early fetal pituitary, i.e., positive staining for alpha-MSH and the presence of ACTH in PNS and cisternae of RER.     

Light and electron microscopic localization of ATPase in normal and degenerating testes of Syrian hamsters.

The distribution of Mg++-activated ATPase was determined with light and electron microscopy in normal and degenerating seminferous tubules. In the normal animals ATPase was localized in the interface between spermatids and Sertoli cells, in association with the cytoplasmic filaments contained within Sertoli cell processes, and in the lymphatic endothelium. ATPase activity increased in degenerating tubules as observed by light microscopy. Electron microscopic investigations of the degenerating tubules which contained only spermatogonia and Sertoli cells revealed reaction product on the outer surface of the Sertoli cell processes and within the interface between adjacent Sertoli cells. Reactaction product was also observed in the Sertoli cell processes between the cytoplasmic filaments and the cell membrane. Where filaments were absent in Sertoli cell processes, no reaction product was observed. These electron microscopic studies indicate that the increase in ATPase activity in testicular degeneration is probably a relative increase due to a loss of the germinal elements of the tubular epithelium and subsequent apposition of the Sertoli cell processes. We speculate that the ATPase activity localized within the Sertoli cell processes may be involved in providing an energy source for filament motility.     

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 microscopic and electron microscopic study of the cells of tissue cultures irradiated with a neodymium laser]

Damage of tissue culture cells (Hela, a human amnion, a primary rat liver culture) caused by the neodymium laser radiation was studied by time-lapse phase-contrast microfilming and electron microscopy. The tested cultures were shown to display different sensitivity and the degree of cell alteration within the same cultures varied considerably. A physical mechanism of cell damage is probably of thermal and shock-wave nature.     

Light and electron microscopic studies of cellular localization of oPL with monoclonal and polyclonal antibodies.

Accurate knowledge of placental lactogen localization is fundamental to any hypothesis of its synthesis and secretion. We used locally generated monoclonal and polyclonal antibodies from three separate sources to localize ovine placental lactogen immunoreactivity on light and electron microscope Lowicryl K4M sections of ovine placentomes of 97-145 days of gestation, using immunogold techniques. All antibodies demonstrated that immunoreactivity was exclusively localized in the trophoectoderm binucleate cell Golgi body and granules and in granules in the syncytium derived from binucleate cell migration. No evidence was found to support a recent claim that monoclonal antibodies to oPL that were produced in Canada indicated a predominant localization of ovine placental lactogen to uninucleate trophectodermal cells.     

Light and electron microscopic localization of D-galactosyl residues in capillary endothelial cells of the canine cerebral cortex

Ricinus communis agglutinin I (RCA-I), a lectin that binds to D-galactosyl residues, intensely stained capillaries in cryostat sections of canine cerebral cortex when evaluated by the avidin-biotin-peroxidase complex method. Of seven lectins tested, only RCA-I gave strong staining of vessels and capillaries with little staining of other cortical cells. Ultrastructural studies using ferritin-, biotin-, and peroxidase-labeled RCA-I indicated that this lectin was bound to the luminal membrane of the cerebral capillary endothelial cell and that lectin receptors were distributed continuously along this membrane. Plasmalemma invaginations that bound RCA-I were also present in endothelial cells. Primary cultures of cerebral capillary endothelial cells grown on plastic or gelatin-coated glass substrates demonstrated staining of the cell membrane and perinuclear structures which appeared to be the Golgi complex and secondary lysosomes. These staining characteristics were retained when the cells were subcultured and were confirmed by ultrastructural studies. In contrast, light microscopy showed that fibronectin was more widely distributed in the cytoplasm, a finding consistent with its occurrence in the endoplasmic reticulum. This work provides support for the concept that lectins may be useful endothelial cell markers in both intact tissue and cell culture.