bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell Membrane Polarity in Dissociated Frog Urinary Bladder Epithelial Cells
Authors: Fujimoto Toyoshi , Ogawa Kazuo
Published: April 2011

Localization of cell membrane markers in dissociated frog urinary bladder epithelial cells was studied. The bladder cells began to alter in shape immediately after dissociation and became spherical within 15 min at room temperature. The apical marker, dialyzed iron (DI), was found on the entire surface of dissociated and transformed cells, whereas the basolateral marker, horseradish peroxidase-glutaral-dehyde (HRP-GLA) staining, first gathered at one pole and then became distributed over a larger area. Thus the apparent loss of polarity was not parallel with the true surface uniformity. When bladder cells were dissociated in the presence of cytochalasin B (CB) or 2,4-dinitrophenol (DNP), the transformation was suppressed and the cells maintained structural polarity. DI and HRP-GLA were mostly confined to the original region in CB-treated cells, but both moved to the other membrane region in DNP-treated cells. The results indicated that an aerobic adenosine triphosphate supply, as well as the tight junction, is involved in maintaining regional differentiation of the cell membrane.
What is Known of the Production of Basement Membrane Components
Authors: Laurie G.W. , Leblond C.P.
Published: April 2011

Immunohistochemistry was used to identify basement membrane components and examine their production by associated cells. Four substances were identified in a series of basement membranes in rats aged 20 days to 34 months, namely, type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin. They were then all localized to the basal lamina part of basement membranes and, presumably, are integrated within this layer. The production of type IV collagen was first examined in the embryonic endodermal cells associated with Reichert's membrane in the rat parietal yolk sac. The rough endoplasmic reticulum (rER), Golgi apparatus, and putative secretory granules of endodermal cells were immunostained, suggesting that these organelles participated in the biogenesis of type IV collagen. However, in rats aged 20 days or more, the cells associated with basement membranes were usually unstained. An exception was noted in the continually growing incisor tooth where the endothelial cells at the proliferating end usually showed immunostaining of rER and Golgi apparatus. It is, therefore, proposed that the formation of type IV collagen for basement membrane occurs at an early stage of development in the life of producer cells. Little is known of the formation of other basement membrane components during development, but there is immunohistochemical evidence that laminin and fibronectin are produced along the same secretory pathway as type IV collagen.
Application of the Microcomputer in Multiparametric Fluorescence Cytophotometry
Authors: Fukuda Masaru
Published: April 2011

Methods for combined cytofluorometric determinations of nuclear DNA plus other parameters (proteins, enzyme activity, or immunoreactive substances) are reported. DNA measurement was performed on an autoradiograph of Feulgen-stained fetal mouse hepatocytes. In order to facilitate the multiparametric measurements and data processing, a fluorescence cytophotometer (BH2-QRFL, Olympus, Tokyo) equipped with four interchangeable filter-sets and controlled by a desktop computer (HP-85F), was used. All data stored in the same computer were displayed in a two-dimensional scatter plot or two-dimensional frequency distribution histogram. Analysis of the DNA histogram of fetal mouse hepatocytes was accomplished by the same computer. Calculated fraction sizes for G1, S, and G2 plus M phases were found to be in good correlation with those determined directly on the simultaneously combined tritiated thymidine (3H-TdR) autoradiography.
Rapid Analysis of Normal and Abnormal Cell Types in Human Semen and Testis Biopsies by Flow Cytometry
Authors: Evenson Donald P. , Melamed Myron R.
Published: April 2011

A simple, rapid procedure is described that quantitates RNA content and DNA content/chromatin condensation for each of many possible cell types and differentiation levels of the cells present in human semen. A fresh semen sample (1-6 hr postemission) or frozen sample (allowing samples to be accumulated and sent to a laboratory) is treated with a detergent solution, stained with acridine orange (AO), and measured by flow cytometry (FCM); approximately 10 minutes are required to measure 5,000 cells per sample and analyze the data with computer assistance. The following can be learned from a single measurement: a) the percentage of each cell type in semen including, i) mature sperm, ii) immature sperm precursor cells, representing all stages of development from spermatogonia to mature sperm, iii) somatic cells, e.g., leukocytes; b) normality/abnormality of sperm nuclear chromatin condensation. These measurements can be correlated with cell types in testis biopsies identified by two-parameter FCM measurements (RNA versus DNA) using AO as the fluorescent probe.
Distribution of Serotonergic Neurons in the Central Nervous System: A Peroxidase-Antiperoxidase Study with Anti-Serotonin Antibodies
Authors: Takeuchi Y. , Kimura H. , Matsuura T. , Yonezawa T. , Sano Y.
Published: April 2011

Distribution of serotonin (5-HT) neurons in the central nervous system (CNS) of various vertebrates was investigated with a highly sensitive immunohistochemical technique. Antibodies were raised in rabbits against an antigen prepared by coupling 5-HT to bovine thyroglobulin. 5-HT neurons were found to be distributed more widely and densely than has been heretofore described. Serotonergic neuronal somata are organized according to certain basic patterns, but there are interspecific differences with regard to the distribution of 5-HT fibers. The processes of 5-HT neurons form a dense plexus by ramification and anastomosis in almost all areas of the CNS, including the ventricular surfaces. In the light of our observations, Golgi's reticular theory may have to be revised.
Calcium Detection in Secretion Granules of Avian Oviduct by Scanning Electron Microscopy (SEM) and Energy-dispersive X-Ray Microanalysis (EDX)
Authors: Makita Takashi , Hatsuoka Masanori , Sugi Yukiko
Published: April 2011

Secretion granules in the shell gland, isthmus, and albumin-secreting region of the hen oviduct were analyzed with WET-scanning electron microscopy (SEM) and EDX, a combination of wide-angle backscattered electron detector (BED) and energy-dispersive X-ray microanalyzer (EDX). Glutaraldehyde-fixed but unhydrated, unstained, and uncoated samples were analyzed; Ca was localized in all secretion granules in all three sections of the hen oviduct studied.
Effect of cisplatin on the plasma membrane phosphatase activities in ascites sarcoma-180 cells: a cytochemical study.
Authors: Aggarwal S K , Niroomand-Rad I
Published: April 2011

To study the effects of cisplatin [cis-dichlorodiammine-platinum (II)] on tumor cells in the presence or absence of the immune system, animals with ascites sarcoma-180 tumor burden were treated with therapeutic dose levels (9 mg/kg). Similarly, ascites sarcoma-180 cells were maintained in tissue culture media containing the same levels of the drug. Cell samples were taken from the animals at 12-hr intervals for 3 days, whereas samples were drawn from the tissue cultures at 15-, 30-, 45-, and 60-min and at 2-, 3-, 4-, and 5-hr intervals. Treated and untreated cells from in vitro and in vivo experiments, when checked for alkaline phosphatase, 5'-nucleotidase, Ca2+-ATPase, and Na+-K+-ATPase, show a gradual decrease in activity on the plasma membrane. It takes about 60 min for inactivation of any enzyme in vitro, whereas it takes 2 days in in vivo experiments. Quantitative analysis show alkaline phosphatase activity drops from 9.7 to 4.9 nmol in just 15 min, and drops further to 0.79 nmol after 2 hr. Inactivation of various plasma membrane enzymes, resulting in permeability changes, is probably responsible for cell death.
Comparison of fixation and penetration enhancement techniques for use in ultrastructural immunocytochemistry.
Authors: Eldred W D , Zucker C , Karten H J , Yazulla S
Published: April 2011

Electron microscopic immunohistochemistry, although generally providing good localization, has often failed to produce satisfying ultrastructural preservation. Techniques that result in well-preserved tissue ultrastructure often hinder penetration of immunological reagents or render antigens non-immunoreactive. These are particularly serious limitations in studies of central nervous system and retina. We have evaluated several fixatives, including picric acid, high pH paraformaldehyde, and glutaraldehyde with subsequent sodium borohydride treatment, and penetration enhancement techniques, including buffered-ethanolic treatment and freeze--thaw, for their applicability in the retina. Our best fixation was achieved with 1 hr in 4% paraformaldehyde and 0.2% glutaraldehyde (pH 7.4) followed by overnight fixation in 4% paraformaldehyde (pH 10.4). Treatment with sodium borohydride after glutaraldehyde fixation restores much of the immunoreactivity that would otherwise be undetectable. The penetration of immunological reagents can be greatly increased by using either a buffered-ethanolic series or by freezing the tissue after careful cryoprotection. Using these methods we have been able to achieve specific immunological staining throughout the full thickness of retinal slices, up to 500 microns across, while preserving good ultrastructure. The methods should prove useful in the immunocytochemical localization of many different antigens in a variety of tissues.
Quantitative cytochemistry of the diaminobenzidine cytochrome oxidase reaction product in mitochondria of cardiac muscle and pancreas.
Authors: Perotti M E , Anderson W A , Swift H
Published: April 2011

The rate constant (k) of the cytochrome oxidase reaction under optimal conditions for cytochemical staining (i.e., 15 min fixation, incubation for 180 min for heart, 120 min for pancreas) can be used as a measure of the enzyme concentration within mitochondria. The rate constant derived from microdensitometric measurements of the mass thickness of the 3,3'-diaminobenzidine (DAB) cytochrome oxidase reaction in cristae times correlated data derived from morphometry on the surface density of cristae (SVcristae/Vmit micron-1) and the volume density of mitochondria per cell (Vmit/Vcell) has been used to determine the respiratory index (RI) of these tissues according to the following equation: RI = k(SVcristae/Vcell). Using this formula, the RI of cardiac muscle tissue was computed to be 33 times the RI of pancreas under the conditions of our experiments. The greater cristae surface density and the large mitochondrial volume density in cardiac muscle and high k value accounted for the higher RI of cardiac muscle.
Light microscopic histochemical detection of sugar residues in secretory glycoproteins of rodent and human tracheal glands with lectin-horseradish peroxidase conjugates and the galactose oxidase-Schiff sequence.
Authors: Schulte B A , Spicer S S
Published: April 2011

Lectins conjugated to horseradish peroxidase were used to stain paraffin sections of mouse, rat, and human respiratory tract tissues. An additional method was applied utilizing galactose oxidase to oxidize the C-6 hydroxyl of galactose and N-acetylgalactosamine residues and the resulting aldehyde was visualized with 2% Schiff's reagent. Sections were stained prior to and after removal of sialic acid residues. Oligosaccharides with terminal beta-galactose and alpha-N-acetylgalactosamine residues were found in all serous cells in the mouse trachea but were never seen in human tracheal serous cells. About 5-10% of serous cells in the rat trachea contained terminal beta-galactose, whereas all human tracheal serous cells and the remainder of those in the rat contained oligosaccharides with terminal sialic acid and penultimate beta-galactose residues. Fucose was not detected in tracheal serous cells of any species. Mucous cells in the mouse and rat trachea produced heterogeneous oligosaccharides containing terminal alpha-N-acetylgalactosamine and/or terminal sialic acid residues in various proportions. The structure of oligosaccharides in human tracheal mucous cells varied between individuals and was related to ABO blood group reactivity. The majority of oligosaccharides in type A individuals contained terminal alpha-N-acetylgalactosamine, whereas type AB individuals had approximately equal amounts of terminal alpha-galactose and alpha-N-acetylgalactosamine residues. Mucous cells in the two type O specimens examined contained a large amount of terminal beta-galactose and surprisingly, terminal fucose was not detected in these individuals. These results support biochemical studies showing structural diversity in oligosaccharide chains of respiratory tract secretions and reveal differences in glycoprotein secretion of different cell types.
Vascular permeability in the rat eye to endogenous albumin and immunoglobulin G (IgG) examined by immunohistochemical methods.
Authors: Pino R M , Thouron C L
Published: April 2011

Vascular permeability in the rat retina and choroid was examined by localizing endogenous albumin (radius, 35 A) and immunoglobulin G (IgG; radius, 55 A) by immunohistochemistry. Three techniques were used: protein A-horseradish peroxidase, peroxidase-antiperoxidase, and avidin-horseradish peroxidase. The protein A-horseradish peroxidase method yielded the least amount of tissue background staining with a high degree of reaction product found in blood vessels. With this method albumin was identified in retinal capillaries, the choriocapillaris, larger choroidal vessels, and in the stroma of the choroid. Very low levels were found in Bruch's membrane. Reaction product due to IgG was also present intravascularly, but little reaction product was present around the large vessels of the choroid and none was identified in Bruch's membrane. Comparisons were made between these localizations and those of intravenously injected hemeprotein tracers of similar size.
Applications of immunocolloids in light microscopy. II. Demonstration of lectin-binding sites in paraffin sections by the use of lectin-gold or glycoprotein-gold complexes.
Authors: Roth J
Published: April 2011

A new procedure is presented for the light microscopic demonstration of specific sugar sequences of oligosaccharides in glycoconjugates by lectins combined with the colloidal gold marker system. Tissue sections from aldehyde-fixed and paraffin embedded rat kidney were stained either in a one-step method with lectin directly bound to particles of colloidal gold or in a two-step method using non-labeled lectin and glycoprotein labeled with colloidal gold. In both methods the presence of lectin-binding sites in the tissue sections is revealed by the appearance of a red coloration that is due to the accumulation of gold particles. The high specificity of the technique is combined with a good sensitivity and resolution as demonstrated by a differential plasma membrane staining in renal epithelial cells. The lectin-gold or glycoprotein-gold complexes remain stable for months and produce a permanent nonbleaching staining.
Immunohistochemical localization of collagenase in hepatic murine schistosomiasis.
Authors: Biempica L , Takahashi S , Biempica S , Kobayashi M
Published: April 2011

It has been previously demonstrated that collagenase activity and collagen synthesis in hepatic granulomas of mice infected with S. mansoni cercariae are maximal 8 weeks after infection; however, total liver collagen content continues to increase. Now the anatomic relationships among collagenase and collagen, granulomas, and hepatic parenchyma in normal mice and in mice infected with S. mansoni are studied. Trypsin-activated collagenase was purified from the media of cultured granuloma explants and anti-collagenase immunoglobulin G was purified from immunized rabbits. The IgG cross-reacted with liver granulomas and active and inactive forms of collagenase, but did not react by immunodiffusion in agar with other neutral proteases or homogenates of schistosome eggs or normal liver. Cryostat sections of liver from normal and infected mice were studied by indirect immunohistochemical methods using fluorescein, rhodamine, and peroxidase labels. Collagenase localization was restricted to areas of collagen deposits in granulomas and hepatic parenchyma. Ultrastructural studies revealed collagenase on the surface of collagen fibers. Hepatocytes of normal mice showed delicate staining at the sinusoidal surface. At all times, immunoreactive collagenase was intimately associated with its substrate, where it presumably initiated collagen degradation. This localization provides a rationale for possible therapeutic approaches to control fibrogenesis through collagenase induction or activation.
Serotonin-like immunoreactivity and anti-5-hydroxytryptamine (5-HT) antibodies: ultrastructural application in the central nervous system.
Authors: Brusco A , Peressini S , Pecci Saavedra J
Published: April 2011

Serotonin immunoreactivity of rat neurons was studied using antibodies to 5-hydroxytryptamine (5-HT) raised in rabbits and the peroxidase-antiperoxidase (PAP) technique. The brains were fixed by perfusion with 4% paraformaldehyde + 0.25% glutaraldehyde. Vibratome sections were incubated in 1:1,000 to 25,000 antiserum dilutions. Specificity of 5-HT immunoreactivity was investigated following immunochemical and immunocytochemical criteria. Ultrastructural analysis of the raphe neurons allowed intracellular localization of 5-HT immunoreactivity in somata, dendrites, and synaptic terminals. Granular reaction product deposits were found attached to microtubules, endoplasmic reticulum membranes, synaptic vesicles, and outer mitochondrial membranes. In all cases the reaction product was confined within the limits of the plasma membrane. It is proposed that binding of 5-HT molecules to tissue proteins via a Mannich reaction and formation of intramolecular condensation products (fluorophores) result from aldehyde fixation. The immunogen (5-HT-bovine serum albumin (BSA) complex) exhibits spectrofluorometric emission and excitation maxima similar to those previously described for the 5-HT fluorophore studied in protein films and in fixed tissue. It is concluded that our antibody detects 5-HT-like immunoreactivity preserved in the tissue with the configuration of the fluorophore (i.e., 3,4-dihydrocarboline). Close agreement between the results and previous studies of 5-HT neurons with other techniques (formaldehyde-induced fluorescence, biochemical assays) support the potential value of this antibody.
Use of monoclonal antibodies for the localization of tissue isoantigens A and B in transitional cell carcinoma of the upper urinary tract.
Authors: Chapman C M , Allhoff E P , Proppe K H , Prout G R
Published: April 2011

Employing the indirect immunoperoxidase technique, monoclonal antisera against blood group antigens A and B were used to localize the corresponding tissue isoantigens in normal ureter and transitional cell carcinoma of the ureter and renal pelvis in 29 patients. All five cases of normal ureters showed positive staining of tissue isoantigens within the transitional epithelium, and all twelve cases of noninvasive transitional cell carcinoma showed similar staining in tumor cells. Of the remaining twelve cases who had invasive tumor, eight lacked tissue isoantigens, while four cases exhibited positive staining. These results support the earlier findings that normal urothelium and noninvasive transitional cell carcinoma of the urinary tract possess ABO tissue isoantigens, while these isoantigens are most frequently absent in invasive tumors. In addition, this study also demonstrates that invasive transitional cell carcinoma of ureters and renal pelvis may continue to possess tissue isoantigens when studied by this sensitive, specific method.
Observations on the cytochemistry of the hemocytes of an insect, Galleria mellonella.
Authors: Chain B M , Anderson R S
Published: April 2011

A number of cytochemical parameters of the hemocytes of larval Galleria mellonella, an insect frequently used as a model by comparative cellular immunologists, are described. Cytochemical methods were used to quantify hemocyte granule-associated components, the results are compared to those obtained for leukocytes from higher animals. Granulocytes contained a population of nonlysosomal granules rich in mucopolysaccharide not seen in plasmatocytes. The numbers and dimensions of these granules showed a positive correlation to cell size, probably reflecting a developmental sequence in granulocyte maturation. Both granulocytes and plasmatocytes had other granules containing the typical lysosomal enzymes, acid phosphatase, beta-glucuronidase, esterase, and lysozyme. The nonlysosomal enzyme alkaline phosphatase was not found in Galleria hemocytes; it is also absent from vertebrate monocytes, macrophages, and immature polymorphonuclear leukocytes. Insect hemocytes appear to lack certain components of antibacterial systems typical of mammalian blood cells, such as H2O2-generating systems, cationic proteins, and myeloperoxidase. The bactericidal mechanisms of hemocytes probably involve lysozyme, as well as other biologically active cellular and humoral factors unique to insects.
The use of lead as an ionic tracer for investigating routes of passive fluid transfer.
Authors: Simson J A , Dom R M
Published: April 2011

Several inorganic ions, including lead, barium, silver, and thallium, have been tested as possible tracers for demonstrating fluid-accessible channels in functional epithelia at the ultrastructural level. The most useful of the ionic tracers examined was the lead (plumbous) ion, administered for short time intervals (less than 2 min) and "captured" with phosphate used as the buffer in the fixative. Passive fluid and ion-accessible channels of rat parotid salivary gland have been examined with this method. At short tracer infusion times (0.5-1.0 min), localization of the tracer was primarily extracellular, although intracellular deposits were observed in the following sites: smooth membrane-delimited endocytic vesicles of both epithelial and connective tissue cells, inner Golgi cisternae, and occasional cisternae of rough endoplastic reticulum. The lead tracer readily penetrated tight junctions between parotid acinar cells but rarely passed through the tight junctions between intercalated duct cells and did not penetrate junctions of striated duct cells. Fat cells observed in the stroma of this gland were the only cells that exhibited lead tracer in the cytosol, suggesting that the plasmalemma of this cell type is more permeable to exogenous ions than the plasmalemma of other cell types present in this gland.
Localization of human prealbumin in choroid plexus epithelium.
Authors: Aleshire S L , Bradley C A , Richardson L D , Parl F F
Published: April 2011

Cerebrospinal fluid (CSF) is generally considered to be derived from plasma through a combined process of ultrafiltration and secretion by the choroid plexus. However, the mechanisms ultimately responsible for achieving the final protein composition of CSF are uncertain. Some proteins, in particular prealbumin, are present in quantities not easily explained by usual physicochemical considerations. To investigate the possibility of de novo synthesis by choroid epithelium, we have examined human choroid plexus an ependyma for the presence of prealbumin. Using the peroxidase-antiperoxidase method at the light and electron microscopic level, as well as immunofluorescence, we localized prealbumin in choroid epithelial cytoplasm on the endoplasmic reticulum and in association with the Golgi apparatus. Ependymal cells and stroma did not reveal immunocytochemical labeling. These findings indicate that the choroid plexus epithelium contributes to the final CSF composition by de novo protein synthesis.
Alkaline phosphatase biosynthesis in the endoplasmic reticulum and its transport through the Golgi apparatus to the plasma membrane: cytochemical evidence.
Authors: Tokumitsu S , Fishman W H
Published: April 2011

Enzyme induction of HeLa cell placental alkaline phosphatase with various agents such as prednisolone, sodium butyrate, hyperosmolality (NaCl), or combination of these inducers resulted in the appearance of enzyme activity in the rough endoplasmic reticulum, nuclear envelope, Golgi apparatus, and plasma membrane. In the Golgi apparatus, intense reaction product deposits tended to be concentrated on its trans side, with small vesicles and granules also being positively stained. Inhibition of protein synthesis with cycloheximide was followed by the disappearance of enzyme activity from these cytoplasmic organelles but not from the plasma membrane. Treatment with monensin, a secretory protein transport inhibitor, uniformly increased activity in the rough endoplasmic reticulum while causing marked dilatation of the intensely positive Golgi cisternae. These results suggest that intracellular alkaline phosphatase is newly synthesized in the endoplasmic reticulum and then passes en route through the Golgi apparatus to the plasma membrane. Accordingly, the present system could represent the biosynthesis, transport, and incorporation of the model cell surface enzyme protein to add to the vesicular stomatitus virus glyco-1 (VSV-G) protein and acetylcholine receptor model systems for studying the dynamics of cell surface protein genesis, transport, and membrane integration.
Ultrastructural visualization of complex carbohydrates in epiphyseal cartilage with the tannic acid-metal salt methods.
Authors: Takagi M , Parmley R T , Denys F R , Kageyama M
Published: April 2011

The present study has ultrastructurally applied the tannic acid-ferric chloride (TA-Fe) and the TA-uranyl acetate (TA-UA) methods to thin sections of glutaraldehyde-fixed, unosmicated embedded epiphyseal cartilage from rat tibiae to demonstrate complex carbohydrates. The strongest TA-Fe and TA-UA staining was observed after fixation of the specimens in glutaraldehyde containing TA. TA-Fe (pH 1.5) strongly stained matrix granules presumed to be proteoglycan monomers and chondrocyte secretory granules at various maturational stages but did not stain collagen fibrils and glycogen. TA-UA (pH 4.2) strongly stained matrix granules, intracellular glycogen, and chondrocyte secretory granules, and moderately stained collagen fibrils in the cartilage matrix. Ribosomes and nuclei were not stained above background staining with UA alone. In alpha-amylase-digested specimens, all TA-UA-reactive cytoplasmic glycogen was selectively removed. Testicular hyaluronidase digestion of specimens selectively removed TA-UA staining in matrix granules and all TA-Fe staining. When the pH of the UA solution was reduced to 1.5, TA-UA staining of glycogen and collagen was markedly decreased or absent, whereas staining of anionic sites was unaltered and significantly greater than with UA staining alone. Thus the TA-metal salt methods are pH dependent and allow differential intracellular and extracellular localization of complex carbohydrates in cartilage tissues at the electron microscope level.
A method for the identification of human peripheral blood T lymphocytes by sequential immunogold and esterase double staining.
Authors: Bergroth V , Konttinen Y T , Reitamo S
Published: April 2011

A double staining method involving the sequential use of monoclonal OKT hybridoma antibodies applied in the colloidal immunogold method and followed by a simultaneously capturing azo dye method for the detection of acid alpha-naphthyl acetate esterase (ANAE) is described. Mononuclear leukocytes isolated from human peripheral blood using a Ficoll-Hypaque density gradient were stained. M-pattern ANAE-positive monocytes (diffuse staining) were excluded from the lymphocyte counts. 80 +/- 5% of all lymphocytes were T-pattern ANAE positive (dot-like staining) and 77 +/- 3% were OKT3 positive. 86 +/- 6% of all ANAE-positive T-pattern lymphocytes were also OKT3 positive, and 89 +/- 6% of all OKT3-positive lymphocytes were also ANAE positive. This indicates that ANAE is a good marker for total human T lymphocytes. 53 +/- 10% of human peripheral blood lymphocytes were OKT4 positive and 87 +/- 8% of all OKT4-positive lymphocytes were also ANAE positive. 30 +/- 6% of all lymphocytes were OKT8 positive, and only 26 +/- 18% of all OKT8-positive lymphocytes were ANAE negative. This indicates that ANAE cannot be used to distinguish T-helper and T-suppressor lymphocytes as identified by monoclonal antibodies.
Whole body autoradiographic distribution of exogenously administered renin in mice.
Authors: Iwao H , Nakamura N , Ikemoto F , Yamamoto K
Published: April 2011

The distribution of exogenously administered renin was investigated using whole body autoradiography. Purified renin from mouse submaxillary gland (SR) was labeled with radioactive iodine (125I). This labeled renin (125I-SR) and Na125I were administered into the tail vein of male ddY mice, in doses of 10.2 and 16.4 mu Ci/30 g body weight, respectively. Mice were killed by an overdose of ether, and autoradiography was performed on whole body sections. To separate free 125I liberated from 125I-SR, sections were treated with perchloric acid. A major accumulation of 125I-SR, acid-insoluble, was evident in the renal cortex, whereas the hepatic accumulation of 125I-SR was minor. Radioactivity in the thyroid and submaxillary glands, in the stomach, and in urine was also apparent, but disappeared after acid treatment, except in the thyroid glands. Radioactivity in the brain, intestinal content, spleen, and adrenal glands was nil. These autoradiograms provide the first evidence that exogenously administered renin is mainly distributed in the renal cortex.
Immunohistochemical localization of two proteinases in skeletal muscle.
Authors: Stauber W T , Fritz V , Dahlmann B , Reinauer H
Published: April 2011

Immunohistochemical localizations of cytosolic and myofibrillar proteinases revealed a different myofiber locale for each enzyme in the rat. Although the cytosolic proteinase was most pronounced in mast cells within soleus and extensor digitorum longus (EDL) muscles, certain fibers of the EDL were also positive. The myofibrillar proteinase, on the other hand, appeared to be present in interstitial spaces between muscle fibers in the EDL but conversely present in some fibers of the soleus muscle.
Immunological characterization of an anti-actin antibody specific for cytoplasmic actins and its use for the immunocytological localization of actin in Aplysia nervous tissue.
Authors: Lubit B W , Schwartz J H
Published: April 2011

Previous immunochemical and immunocytochemical studies have shown that an antibody to actin prepared from body wall muscle of the marine mollusc Aplysia californica is specific for vertebrate cytoplasmic actins. The ability of this anti-actin to distinguish between different forms of actin most likely reflects the recognition of amino acid sequences unique to cytoplasmic actins. We have confirmed the specificity of this antibody for cytoplasmic actins using nervous tissue as a source of cytoplasmic actin in further immunochemical studies. In addition to binding cytoplasmic actin in purified preparations, the antibody removed actin selectively from crude extracts of nervous tissue of some but not all of the species tested. Our results also suggest that tissue-specific differences in the distribution of cytoplasmic actins may exist. Immunofluorescence studies of Aplysia nervous tissue stained with anti-actin revealed that actin is present in the cell body and axonal processes of Aplysia neurons. Although the function of actin in nerve cells is not understood, the observed pattern of immunofluorescence staining is consistent with the idea that actin may be involved in movement within the axoplasm.
Effect of monensin on secretory pathway in GH3 prolactin cells. A cytochemical study.
Authors: Tougard C , Picart R , Morin A , Tixier-Vidal A
Published: April 2011

The effects of the carboxylic ionophore monensin have been studied on a rat prolactin cell line (GH3 cells) under basal conditions or after acute stimulation by thyrotropin-releasing hormone (TRH). It was found that 1) monensin induces a rapid dilatation of Golgi elements in these endocrine cells; 2) secretory product, prolactin, is localized by electron microscope immunocytochemistry attached to the inner face of the membrane of these dilated vacuoles; 3) monensin induces preferentially a dilatation of the cis face of the Golgi zone, since the "GERL" complex identified by acid phosphate cytochemistry is disorganized or fragmented rather than vacuolized; and 4) monensin decreases strongly the basal release of prolactin in the culture medium but does not prevent the stimulating effect of TRH on this release. This suggests that monensin blocks preferentially the pathway of release of secretory product under basal conditions in GH3 cells but that another pathway less sensitive to monensin is involved under acute stimulation by TRH.
Tritium autoradiography of cell surfaces in smear preparations.
Authors: Dörmer P , Jäger G
Published: April 2011

Experiments were performed in order to find out whether tritium-labeled cell surface markers can be quantified at the single cell level in autoradiographs of smear preparations. Mouse thymocytes were incubated with 3H-concanavalin A and subsequently spread on microscopic slides. The spreading techniques, either by cytocentrifugation or by the use of cover slips, were performed in such a way as to achieve preparations in which the mean flatness of the cells varied. By means of incident light microphotometry, the cellular areas and the grain counts of individual cells were determined. The results show a strong dependence of the mean grain yield per slide on the mean cellular area. Cytocentrifuge preparations resulted in larger mean cellular areas and higher mean grain counts than cover slip preparations. With the use of cytocentrifugation, however, the differences in the flatness of the cells were of such a magnitude that a reproducible quantification of 3H-labeled cell surface markers was not possible. Conventional techniques of spreading cells on slides failed to provide a degree of flatness that could approach the saturation grain count per cell without completely destroying the cellular morphology.
The influence of embedding media and fixation on the postembedment ultrastructural demonstration of complex carbohydrates. III. High iron diamine staining for sulfated glycoconjugates.
Authors: Thomopoulos G N , Schulte B A , Spicer S S
Published: April 2011

The high iron diamine (HID) method for detection of sulfated complex carbohydrate has been applied directly on thin sections of variably fixed tissues embedded in epoxy and nonepoxy resins. Results with postembedment HID staining in mouse intestinal epithelium are compared to those previously obtained using preembedment methods. Sections from epoxy-embedded tissues have been found to exhibit the weakest staining intensity. Intense, specific staining was obtained in tissues not postfixed with osmium tetroxide and embedded in polystyrene, polyester resins, styrene-methacrylate, and especially the styrene-Vestopal W embedding mixture. Postosmication of tissues abolished HID staining in epoxy resins and the styrene-Spurr's resin embedding mixture, but only reduced the staining intensity in tissues embedded in nonepoxy resins.
Rapid method for identification of macrophages in suspension by acid alpha-naphthyl acetate esterase activity.
Authors: Ennist D L , Jones K H
Published: April 2011

A supravital staining procedure for the identification of macrophages in cell suspension using a modification of a standard cytochemical assay for alpha-naphthyl acetate esterase (ANAE) activity is described. Macrophages are stained an intense red-brown after 5 min incubation in a buffer using ANAE as the substrate and hexazonium pararosaniline as the coupler for the azo dye. There is close agreement in the number of ANAE-positive cells found and the number of macrophages identified in smears by morphological criteria, by phagocytosis, and by the presence of Fc receptors. Therefore, this stain provides a quick, inexpensive method to estimate the number of macrophages present in suspensions of lymphocytic tissues from rats and mice.
Double immunogold staining method for the simultaneous ultrastructural localization of regulatory peptides.
Authors: Tapia F J , Varndell I M , Probert L , De Mey J , Polak J M
Published: April 2011

Recent studies have suggested that the morphological characteristics of secretory granules contained within endocrine cells and nerves may be determined largely by their chemical composition. The use of the immunogold staining (IGS) method, which is based on the adsorption of colloidal gold to immunoglobulins, has been used in our laboratory to demonstrate a wide range of intracellular antigens at both the light and electron microscope levels. In this study we have applied a modification of the IGS method for the simultaneous detection of two separate antigens in a single tissue section, using a variety of region-specific antisera to different peptides. Peptide antisera were raised in rabbits or in guinea pigs and these were applied simultaneously or sequentially to grid-mounted ultrathin tissue sections. Antigenic sites were visualized at the electron microscope level using antisera raised in goats, adsorbed to gold particles of 12, 20, or 40 nm. Using this technique we have attempted to investigate the coexistence of multiple antigens in single tissue sections, in particular in single granules; the topographic distribution of molecular forms within one single granule or granule population; the heterogeneity of peptidergic neurons and also the heterogeneity of peptide content in morphologically similar granules. The double immunogold staining procedures described here have proved to be extremely effective for the simultaneous ultrastructural localization of two antigens (peptide-peptide; peptide-propeptide) on a single tissue section. The further development of this technique may provide useful information on neuroendocrine cell dynamics in normal and diseased states.
The interaction of 125I-insulin with cultured 3T3-L1 adipocytes: quantitative analysis by the hypothetical grain method.
Authors: Fan J Y , Carpentier J L , Van Obberghen E , Blackett N M , Grunfeld C , Gorden P , Orci L
Published: April 2011

The murine 3T3-L1 fibroblast under appropriate incubation conditions differentiates into an adipocyte phenotype. This 3T3-L1 adipocyte exhibits many of the morphologic, biochemical, and insulin-responsive features of the normal rodent adipocyte. Using quantitative electron microscopic (EM) autoradiography we find that, when 125I-insulin is incubated with 3T3-L1 adipocytes, the ligand at early times of incubation localizes to the plasma membrane of the cell preferentially to microvilli and coated pits. When the incubation is continued at 37 degrees C, 125I-insulin is internalized by the cells and preferential binding to the villous surface is lost. With the internalization of the ligand, two intracellular structures become labeled, as determined by the method of hypothetical grain analysis. These include large clear, presumably endocytotic, vesicles and multivesicular bodies. Over the first hour of incubation the labeling of these structures increases in parallel, but in the second hour they diverge: the labeling of multivesicular bodies and other lysosomal forms continuing to increase and the labeling of large clear vesicles decreasing. At 3 hours limited but significant labeling occurs in small Golgi-related vesicles that have the typical distribution of GERL. The distinct morphologic features of this cell make it ideal for a quantitative morphologic analysis and allow for an unambiguous view of the sequence of events involved in receptor-mediated endocytosis of a polypeptide hormone. These events are likely to be representative of the processing of insulin by the mature rodent adipocyte.
Regional distributions of choline acetyltransferase and acetylcholinesterase activities in layers of rat cerebellar vermis.
Authors: Ross C D , Smith J T , Godfrey D A
Published: April 2011

Entire sagittal sections of rat cerebellar vermis were dissected into microgram-sized samples of molecular, granular, and white matter layers. Assayed activities of choline acetyltransferase and acetylcholinesterase were plotted back onto sectional maps of exact sample locations. On the average, the activities of choline acetyltransferase and acetylcholinesterase in the granular layer were about four and five times, respectively, those in the molecular layer. The highest activity of both enzymes was in the granular layer of the vestibulocerebellum, the nodulus and ventral uvula (lobules X and IXc of Larsell). This activity might be related to the secondary vestibulocerebellar projection, terminating as mossy fibers in the granular layer of this region. Intermediate levels of activity were found in the granular layer of the dorsal uvula (lobule IX, a and b). The lowest activities of both enzymes in the granular layer were in the culmen (lobule V). A 7.1-fold difference in choline acetyltransferase activity and a 4.5-fold difference in acetylcholinesterase activity were found between the granular layer of lobules V and X. The numerical density of aggregates of acetylcholinesterase staining product in the granular layer correlated much better with assayed acetylcholinesterase activity than with choline acetyltransferase activity.
Differential filipin labeling of the luminal membranes lining the pancreatic acinus.
Authors: Orci L , Perrelet A , Montesano R
Published: April 2011

The cytochemical labeling of cholesterol by filipin in plasma membranes of the exocrine pancreas has shown a difference in the reactivity to filipin between the acinar and centro-acinar cells. The acinar cell has a poorly labeled luminal membrane but numerous filipin-cholesterol complexes on the basolateral membrane. In contrast, the centro-acinar cell shows a comparable degree of filipin labeling on both luminal and basolateral membrane. This difference in the distribution of filipin labeling suggests underlying differences in the organization of the respective membranes, probably related to their specific functions.
Immunohistochemical staining on hydroxyethyl-methacrylate-embedded tissues.
Authors: Casanova S , Donini U , Zini N , Morelli R , Zucchelli P
Published: April 2011

Hydroxyethyl-methacrylate (GMA) embedding has recently been proposed for light microscopy studies. In the present investigation extracellular protein antigens were localized on GMA-embedded renal biopsy tissue. Conventionally frozen sections were compared with GMA sections from 55 renal specimens for the detection of extracellular protein antigens. Sections were directly stained with fluorescein- or peroxidase-conjugated antisera against immunoglobulin (Ig) G, IgA, IgM, C3, C1q, and fibrinogen. Results obtained using these two methods showed a 74-89% agreement, depending on the antigen under study. Some discrepancy between GMA and frozen sections was observed in three cases of renal amyloidosis and those cases presenting focal or trace reactions; the differences did not, however, influence the diagnosis. Prerequisites for antigen recovery on GMA sections were a) choice of fixative; b) abrupt dehydration of specimens; and c) treatment of sections with nonspecific protease. The improved localization and the lower background staining obtained led to easy and immediate detection of antigens on GMA sections despite the reduced antigenicity due to the embedding process.
Mechanism of glycosylation in the Golgi apparatus.
Authors: Fleischer B
Published: April 2011

A major role of the Golgi apparatus in liver is the terminal glycosylation of secreted serum proteins and of plasma membrane glycoproteins. Galactosyltransferase is a membrane-bound Golgi enzyme that transfers galactose directly from uridine diphosphogalactose (UDP-Gal) to terminal N-acetylglucosamine groups of N-asparagine-linked glycoproteins during secretion. Sialytransferase then transfers sialic acid from cytidine monophosphosialic acid (CMP-NAN) to the newly added terminal galactose of the glycoprotein. In the cell, the transfer reaction must occur on the lumen side of the Golgi membrane. UDP-Gal is synthesized mainly in the cytoplasm and CMP-NAN is synthesized in the nucleus in liver. An important question for understanding the mechanism is, how do these nucleotide sugars gain access to the transferases? A second question involves uridine diphosphate (UDP), a highly inhibitory product of galactosyltransferase. How is UDP removed from the lumen of the Golgi fast enough to prevent product inhibition of the galactosyltransferase? We have shown that isolated Golgi, although vesiculated, retains its original orientation. The vesicles are oriented with greater than 90% of both galactosyltransferase and sialyl-transferase on the luminal side of the vesicles. Using intact vesicles, we can show that UDP-Gal is taken up via a saturable carrier system present in the Golgi membrane. During galactosylation in vitro, UDP formed in the lumen of Golgi vesicles is rapidly converted to UMP by a nucleoside diphosphatase in the lumen. Uridine monophosphate, which is much less inhibitory to the galactosyltransferase than UDP, is then transported out of the lumen by a second carrier and is broken down further to uridine by 5'-nucleotidase on the cytoplasmic side of the Golgi vesicles. The transport of nucleotides appears unique to the Golgi membranes, since neither rough endoplasmic reticulum nor plasma membrane vesicles from rat liver accumulate these nucleotides.
Ultrastructural cytochemistry and immunocytochemistry of proteoglycans associated with epiphyseal cartilage calcification.
Authors: Takagi M , Parmley R T , Denys F R
Published: April 2011

Proteoglycans (PGs) are closely associated with cartilage calcification. We have examined the hypertrophic zone of rat epiphyseal cartilage, in which calcification is occurring, using the high-iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method for sulfated glycosaminoglycans, an immunoferritin method specific for chondroitin sulfate A, and the tannic acid-ferric chloride (TA-Fe) method to stain cartilage matrix granules (MGs) presumed to be PG monomers. HID-TCH-SP produced stain deposits with a diameter of 11.2 +/- 3.2 nm (mean +/- SD; n = 200) in the MGs. However, HID-TCH-SP staining was not discernible in membrane-limited matrix vesicles (MVs). In areas of advanced calcification, partially disrupted MVs and globular bodies (GBs), derived in part from disrupted and/or degenerated MVs, contained a few too many small HID-TCH-SP stain deposits. Further down the epiphyseal cartilage, intact MVs markedly decreased and the GBs, containing many small HID-TCH-SP stain deposits, significantly increased in number. These GBs were found exclusively in the longitudinal septa rather than in the transverse septa. After enzyme digestion with testicular hyaluronidase, small (7.2 +/- 1.2 nm in diameter) stain deposits remained in the MGs and GBs, presumably localized to keratan sulfate. Immunoferritin localizing chondroitin sulfate strongly stained MGs, whereas MVs and GBs lacked staining. TA-Fe staining of glycoconjugates in the GBs demonstrated a striking decrease in the diameter of MGs associated with calcification in the GBs as compared with those in the noncalcifying area around the GBs. These results indicate that the GBs containing needle-like apatite crystals in morphologic preparations represent sites of chondroitin sulfate degradation. Testicular hyaluronidase-resistant sulfated glycosaminoglycans presumed to be keratan sulfate and partially degraded PGs selectively remain within the GBs as a probable requisite for expansion of the initial calcification in MVs.