bluuebunny/crossref_metadata_2025 huggingface dataset:
Human skeletal muscle fiber type adaptability to various workloads.
Authors: Staron R S , Hikida R S , Hagerman F C , Dudley G A , Murray T F
Published: April 2011

Muscle biopsy specimens were removed from the vastus lateralis muscles of three groups of human subjects: controls, weight lifters, and distance runners. The runners proved to be a unique group with respect to the variables measured (low body weight and percentage body fat, and high VO2 max). Additionally, a histochemical analysis of the biopsy specimens revealed that the runners had a significantly higher percentage of fiber types I and IIC than either the controls or the weight lifters. Using a cryostatic retrieval method, each of the fibers identified histochemically was then analyzed morphometrically using electron microscopy. The results of volume-percent mitochondria demonstrated a strong relationship between the ATPase activity and oxidative potential of the fiber types for all three groups such that the oxidative activity would be ranked I greater than IIA greater than IIB. Irrespective of fiber type, there were significant differences between the groups with regard to muscle-fiber mitochondrial (runners greater than lifters greater than controls) and lipid content (runners greater than controls greater than lifters). The lifters had a significantly greater content of mitochondria than the controls, which may suggest that inactivity rather than the lifting exercise contributes to a low volume-percent mitochondria and a high percentage of type IIB fibers.
The distribution of aggregating proteoglycans in articular cartilage: comparison of quantitative immunoelectron microscopy with radioimmunoassay and biochemical analysis.
Authors: Ratcliffe A , Fryer P R , Hardingham T E
Published: April 2011

Electron microscopic immunolocalization and radioimmunoassay have been used to determine the variation with depth of the hyaluronate-binding region of proteoglycan in articular cartilage. The cartilage was cut into serial sections from the articular surface to the bony margin, the proteoglycans were extracted from each section and determined by radioimmunoassay using antibodies raised against proteoglycan binding region. Proteoglycans were found to be most abundant in the middle zone and least abundant near the articular surface. Biochemical analysis for hexuronate in the same extracts showed a distribution of proteoglycan in agreement with these and other published results. The binding region antiserum was used for electron microscopic immunolocalization of proteoglycan with ultrathin sections of cartilage embedded in Lowicryl K4M resin. After digestion of the sections with chondroitinase ABC, the proteoglycans were localized using the antiserum and protein A-coated gold particles as immunolabel. The density of labeling was quantified using a Magiscan image analysis system. Throughout the depth of the cartilage matrix labeling was higher in the pericellular regions compared to the intercellular regions, and variation of the amount of immunolabel with depth was found to show a good correlation with the results from radioimmunoassay. Intracellular labeling of proteoglycans was mainly found over the Golgi region and in membrane-bound (secretory) vesicles.
Carbohydrate cytochemistry of tracheobronchial airway epithelium of the rabbit.
Authors: Plopper C G , St George J A , Nishio S J , Etchison J R , Nettesheim P
Published: April 2011

Three types of nonciliated secretory epithelial cells contribute to the mucous lining of pulmonary airways: mucous cells, serous cells, and Clara cells. Contrary to observations in other species, airways of the rabbit have very few mucous cells. In the rabbit, the predominant secretory cell throughout the entire airway tree, including the trachea, appears to be one cell type, the Clara cell. While these cells share the same ultrastructural features throughout the tree, the nature of their contribution to the mucous blanket is not clear. This study was designed to characterize the carbohydrate components of secretory granules in tracheal Clara cells, and to compare that carbohydrate with that of tracheal mucous (goblet) cells and with Clara cells of more distal airway generations. Trachea and lungs of six adult male rabbits were fixed by airway infusion, the conducting airways of the right cranial lobe dissected and tissue selected from the trachea and five distal airway generations. For light microscopy (LM), sections of paraffin-embedded tissues were stained with Alcian blue-periodic acid-Schiff (AB/PAS), dialyzed iron (DI), and high iron diamine-Alcian blue (HID-AB). For electron microscopy (EM), fixed tissues were incubated with DI, HID, MgCl2, or buffer, postosmicated, embedded in epoxy resin, and thin sections stained with periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP). By LM, most Clara cells did not react with PAS, AB, HID, or DI. A few in trachea and bronchi had PAS-positive apical margins. Mucous goblet cells were positive with PAS, AB, and HID, indicating sulfated glycoproteins. By EM, a small number of Clara cells had PA-TCH-SP-positive luminal granules, a few luminal granules had DI-positive rims. Almost all Clara cell granules were negative with PA-TCH-SP, HID, and DI. The granules of mucous goblet cells had a finely granular core surrounded by a meshwork of variable density. The meshwork was positive with PA-TCH-SP, DI, and HID. The cores were not. We concluded that: 1) the Clara cell does not contribute carbohydrates to the airway mucous lining; 2) mucous goblet cells secrete predominantly sulfated glycoprotein; and 3) the contribution to mucous carbohydrates by Clara cells does not vary with the airway level in which they are located.
Intraspecific recognition system in scleractinian corals: morphological and cytochemical description of the autolysis mechanism.
Authors: Müller W E , Müller I , Zahn R K , Maidhof A
Published: April 2011

Intraspecific aggressive interactions have been observed in the scleractinian coral Stylophora pistillata. Extensive field observations suggest that intraspecific interactions result in the formation of necrosis in one of the two competing colonies. The formation of necroses is primarily restricted to those layers that are interspersed with polyps. In a second phase the non-living, central corallum is destroyed by foreign microorganisms. In some cases the necroses are resealed again, either by a living septal layer or by a calcareous plug. Evidence is presented indicating that the enzyme carbonic anhydrase has a role in these activities. First, it is involved in the apical growth of the colony. Using a semiquantitative assay procedure, the highest enzyme level was determined in polyps at the tips of the branches; lower levels were measured in polyps of the foot/trunk region. Secondly, the enzyme was found to be induced in those areas that are in close contact with non-isogeneic tissue. From these data we conclude that carbonic anhydrase is involved in the process of autolytic digestion in response to intraspecific contact of two non-clonemates.
Effects of perfusate composition on binding of ruthenium red and gold colloid to glycocalyx of rabbit aortic endothelium.
Authors: Baldwin A L , Winlove C P
Published: April 2011

The effects of perfusate composition on some of the properties of the endothelial glycocalyx were investigated electron microscopically. Rabbit aorta was perfused in situ in preparation for fixation with 1% OsO4 containing 0.1 Ruthenium red (RR) or perfusion with wheat germ agglutinate (WGA)-coated gold particles suspended in 0.9% NaCl, Tyrode's solution, or pooled rabbit serum. Intense RR staining of the intimal glycocalyx was found only in vessels that had been exposed to 150 mmol/L NaCl, pH 7.4, or to albumin (4.0 g/100 ml), gamma-globulin (0.7 g/100 ml), or fibrinogen (0.3 g/100 ml) in Tyrode's solution, pH 7.4, for 5 min prior to fixation. The adherence of WGA-coated gold particles to the intimal and vasa vasorum glycocalyces was found in saline- and Tyrode's-perfused vessels. The density of the WGA-coated gold particles was greatest in the glycocalyx of damaged endothelium. In vessels flushed with serum, no RR stain or WGA-coated gold colloid was seen in the glycocalyx. This study demonstrates that saline perfusion facilitates the passage of RR and WGA-coated gold particles into the endothelial glycocalyx. We conclude that serum proteins and cell-surface glycoprotein chains act cooperatively to impede the access of some molecules to the endothelial surface.
The ultrastructural localization of two basement membrane components: entactin and laminin in rat tissues.
Authors: Martinez-Hernandez A , Chung A E
Published: April 2011

The localization of two noncollagenous components of basement membranes, laminin and entactin, was determined in rat kidney, muscle, and small intestine using electron immunohistochemistry. In the renal glomerulus anti-laminin antibodies reacted with the basement membrane of peripheral capillary loops and with mesangial matrix. In the peripheral capillary loop laminin was preferentially distributed in both laminae rarae. This was in contrast to anti-entactin that localized in peripheral capillary loops but not in mesangial matrix. Even in the peripheral capillary loops it had a different distribution than laminin. Entactin was found predominantly in the lamina rara interna. In renal tubular basement membranes both antibodies localized throughout the full thickness of the basement membranes, with laminin having a preferential distribution in the lamina rara, whereas entactin was more evenly distributed. In the basement membrane of the duodenal mucosa entactin localized in the lamina densa, whereas laminin was present in both laminae. In skeletal muscle both antibodies had similar localization in all basement membranes. These results demonstrate that entactin is an intrinsic component of basement membranes. They also demonstrate that basement membranes from different tissues have subtle variations in content and/or assembly of the different components. It is likely that these variations may be reflected in different functional properties.
Ultrastructure of mast cell degranulation induced by eosinophil peroxidase: Use of diaminobenzidine cytochemistry by scanning electron microscopy.
Authors: Chi E Y , Henderson W R
Published: April 2011

It has been previously demonstrated that eosinophil peroxidase (EPO) when supplemented with hydrogen peroxide and a halide induces noncytotoxic mast cell degranulation. Using a more highly purified EPO preparation, the ultrastructure of EPO-induced mast cell secretion has been studied using transmission and scanning electron microscopy and freeze-fracture techniques. At relatively low EPO concentrations, secretory changes were comparable to those caused by other mast cell secretagogues. Swollen and less electron-dense granules were seen in intracellular channels, some of which opened to the outside of the cell. EPO stimulation led to bulging of the surface membrane by submembranous granules and formation of pores in the cell surface that also contained fewer villous projections than control cells. During the secretory process, plasma membrane bulges were depleted of intramembranous particles in both the E and P faces of the apical regions of the perigranular and plasma membranes. Higher EPO concentrations caused a marked cytotoxic disruption of the mast cells. Diaminobenzidine cytochemistry was used to detect EPO reaction products on the mast cell surface by scanning electron microscopy; this technique should prove useful in detecting peroxidase reaction products on a variety of target cells.
Cytochemical studies of adenylate cyclase in the choroid plexus and brain vessels of rat and mouse.
Authors: Vorbrodt A W , Szumańska G , Dobrogowska D H
Published: April 2011

Adenylate cyclase (AC) activity was studied in the choroid plexus and brain vessels of rat and mouse using a recently improved cytochemical method (Palkama A, Kaufman HE, Uusitalo R, Uusitalo H, Virtanen J, Huhtaniitty S: J Histochem Cytochem 29:898, 1981). For comparison, the vasculature of skeletal muscle was also examined. Specimens were fixed for 5 min in 1% paraformaldehyde or 1% paraformaldehyde and 0.1% glutaraldehyde supplemented with activators of AC: isoproterenol and 5'-guanylylimidodiphosphate or forskolin. These activators were also present in the "activating solution" and in the incubation medium. For control purposes no activators were used. Various capturing agents were introduced into incubation media: SrCl2, Pb citrate, NdCl3, and CeCl3. Satisfactory results were obtained with both fixatives, both activators, and the capturing agents SrCl2 and Pb citrate. In rat choroid plexus AC activity was localized in the basal and lateral plasmalemma of epithelial cells, in the plasmalemma of cells interposed between epithelium and capillaries and occasionally in pinocytotic vesicles of endothelial cells. In micro-blood vessels of rat brain, the reaction appeared in the luminal or in both luminal and abluminal plasmalemma of endothelial cells and in smooth muscle cells. In skeletal muscle microvessels, reaction product was mainly located in numerous pits and pinocytotic vesicles associated with luminal plasmalemma of endothelial cells. Some reaction in rat cortex was occasionally observed in Golgi saccules of neurons and in postsynaptic densities. In the mouse choroid plexus and brain vessels the reaction was notably less intense, suggesting the existence of species differences in AC activity as revealed by cytochemical technique.
Validity of a histochemical estrogen receptor assay. Supported by the observation of a cellular response to steroid manipulation.
Authors: Lee S H
Published: April 2011

A macromolecular hydrophilic fluorescent estradiol conjugate, 17 beta-estradiol-6-carboxymethyl oxime-bovine serum albumin-fluorescein isothiocyanate, was used as a histochemical reagent to study estrogen-binding substances or putative estrogen receptors in cryostatically frozen sections of the rat uterus. Exogenous 17 beta-estradiol injected subcutaneously was found to induce or increase the synthesis of an estrogen-binding substance (or substances) in the cytoplasm of the luminal epithelial cells of the uterus in immature and adult ovariectomized female rats, whereas progesterone antagonized this effect. No effect was observed when hydrocortisone was substituted for progesterone. These intracellular responses to steroid hormone manipulation accord with the known behavior of estrogen receptors in the rat uterus. The results offer further evidence that this histochemical method is capable of identifying cell types rich in estrogen receptors.
Effects of secretory stimulation on the Golgi apparatus and GERL of rat parotid acinar cells.
Authors: Hand A R , Oliver C
Published: April 2011

The structure and cytochemistry of the Golgi apparatus and GERL of rat parotid acinar cells was studied after in vivo secretory stimulation with isoproterenol. Discharge of mature secretory granules was complete within 1 hr after isoproterenol injection, but immature granules in the Golgi region or near the lumen were not released. At early times (1-5 hr) after isoproterenol, acid phosphatase (AcPase) activity was markedly increased in GERL and immature secretory granules compared to uninjected controls. GERL appeared increased in extent and numerous continuities with immature granules were observed. Reaccumulation of mature secretory granules was first evident at 5 hr, and was almost complete by 16 hr after isoproterenol. Thiamine pyrophosphatase (TPPase) activity, normally restricted to the trans Golgi saccules, was frequently present in immature granules during this time. Narrow cisternae resembling GERL, occasionally in continuity with immature granules, also contained TPPase reaction product. By 16-24 hr after stimulation, the activity and distribution of AcPase and TPPase were similar to control cells. These results demonstrate the dynamic nature of the Golgi apparatus and GERL in parotid acinar cells, and emphasize the close structural and functional relationship between these two structures.
Proteoglycans in arterial smooth muscle cell cultures: an ultrastructural histochemical analysis.
Authors: Chen K , Wight T N
Published: April 2011

The extracellular matrix in cultures of arterial smooth muscle cells has been examined by ultrastructural histochemistry using each of the following cationic dyes: ruthenium red, Alcian blue, acridine orange, and safranin O. All dyes exhibited an affinity for a structural component that was either preserved as a granule with ruthenium red or Alcian blue, or as an extended filament or bottlebrush structure with acridine orange or safranin O. Both granules and filaments were removed when the cultures were pretreated with chondroitinase ABC, an enzyme that degrades the glycosaminoglycan moiety of some proteoglycans. These structural components of the extracellular matrix were not observed when cultures were prepared in the absence of the cationic dyes. Labeling experiments (35S-sulfate) revealed that approximately 40% of the total labeled proteoglycans were lost during routine processing for electron microscopy (i.e., fixation through dehydration). Inclusion of any one of the cationic dyes during fixation reduced the losses to less than 1%. The extended filamentous structure preserved by safranin O and acridine orange resembled the structure of purified proteoglycans prepared from the same cultures and spread on cytochrome c monolayer films. These observations suggest that proteoglycans exist as extended bottlebrush structures within the extracellular matrix, and support the interpretation that the granular deposits observed in the ruthenium red and Alcian blue preparations most likely represent individual proteoglycan monomers that have undergone molecular collapse during processing. In addition, the dyes also exhibited an affinity for chords of fine fibrils that contained small granules and/or filaments. Both the fibrillar material and the associated granular and filamentous structures enmeshed in the fibrils resisted digestion with chondroitinase ABC.
The use of osmium-thiocarbohydrazide-osmium (OTO) and ferrocyanide-reduced osmium methods to enhance membrane contrast and preservation in cultured cells.
Authors: Willingham M C , Rutherford A V
Published: April 2011

The preservation and contrast of membranous structures in cultured cells using various postfixation procedures prior to embedding have been investigated. These include routine OsO4, ferrocyanide-reduced OsO4, osmium-thiocarbohydrazide-osmium (OTO), and ferrocyanide-reduced osmium-thiocarbohydrazide-ferrocyanide-reduced osmium (R-OTO). With standard ethanol-Epon dehydration/embedding techniques, a dramatic improvement in both membrane contrast and preservation of bilayer membrane structure was achieved using preembedding OTO in cultured cells. R-OTO yielded similar enhanced preservation and contrast of membranes. Both of these methods also resulted in an increase in the contrast of diaminobenzidine reaction product from horseradish peroxidase activity, and of lipid droplets and lipoprotein particles. However, R-OTO did not cause the same increase in the density of proteinaceous elements as was seen with the OTO method. Ferrocyanide-reduced osmium alone showed significant advantages for quantitation of immunocytochemistry using ferritin labels with bismuth subnitrate counterstain. These methods should have general usefulness for the preservation of lipid-containing structures in cultured cells.
Elastase as a marker for neutrophilic myeloid cells.
Authors: Kramps J A , van der Valk P , van der Sandt M M , Lindeman J , Meijer C J
Published: April 2011

The immunohistochemical results obtained with antibodies directed against human neutrophil elastase is described. It was found that neutrophil elastase can be used as a specific marker of cells of the neutrophilic lineage. In normal hematopoietic cell preparations, only promyelocytes and more differentiated myeloid cells stain positive for elastase. In acute or chronic myeloid and myelomonocytic leukemia, the same neutrophil myeloid cells stain positive, whereas neoplastic cells of the monocytoid line are negative. Using elastase in conjunction with other markers, it is possible to differentiate easily the involvement of different cell lines in myeloproliferative diseases.
Fibronectin in rat heart: a link between cardiac myocytes and collagen.
Authors: Ahumada G G , Saffitz J E
Published: April 2011

Fibronectin, a glycoprotein that binds to collagen and modifies the adhesion properties and motility of cells in culture, is present in the interstitium of rat hearts. To localize fibronectin more precisely and to assess its relationship to the myocyte and to connective tissue elements, we employed a double antibody technique to label myocardial fibronectin with electron-dense ferritin to permit an ultrastructural analysis. Fibronectin was found to be associated with collagen, and in some cases appeared to link collagen fibers. Fibronectin was also found inserted along the surfaces of cardiac myocytes, connecting these cells to perimyocytic collagen. These ultrastructural relationships imply that fibronectin is a major component of the myocardial interstitium, and may affect myocardial compliance and control the motion of myocytes during the contraction and relaxation of the heart.
Block-staining tissues with potassium ferrocyanide-reduced osmium tetroxide and lead aspartate for electron microscopic radioautography.
Authors: Kopriwa B M
Published: April 2011

In the hope of devising a method for prestaining tissues en bloc for electron microscopic radioautography, pieces of radioiodine-labeled liver were taken through various combinations of ferrocyanide-reduced osmium tetroxide, lead aspartate, and aqueous uranyl acetate at room temperature or at 60 degrees C. Following the tests, the method adopted for routine use was to block-stain tissues for 2 hr in potassium ferrocyanide-reduced osmium tetroxide at 4 degrees C followed by 1 hr in Walton's lead aspartate at room temperature. This simple method, which requires no manipulation before or after emulsion coating and development of the radioautographs, provides adequate contrast without inducing background fog or artifacts.
Immunocytochemical localization and circadian variations of serotonin and N-acetylserotonin in photoreceptor cells. Light and electron microscopic study in the teleost pineal organ.
Authors: Falcon J , Geffard M , Juillard M T , Steinbusch H W , Seguela P , Collin J P
Published: April 2011

Using two immunocytochemical procedures (i.e., immunofluorescence and the unlabeled peroxidase-antiperoxidase method), the localization of a serotonin(HT)-like and of a N-acetylserotonin (aHT)-like immunoreactivity in the pineal organ of the pike was studied during winter. It was shown that immunostaining was exclusively restricted to the cells of the receptor line (CRL = typical and modified photoreceptors). The intensity of the reactions varied through the light-dark cycle, HT-like immunoreactivity being high during the photophase and low during the scotophase. In contrast, aHT-like immunoreactivity was highest at the beginning of the scotophase. HT and aHT-like immunoreactivities were detected in all cell types of the pineal epithelium after administration of a monoamine oxidase inhibitor. Up to now, only HT immunoreactivity could be localized at the ultrastructural level. In a number of typical and modified photoreceptors, a HT-positive staining seemed to be confined within the hyaloplasm of the inner segment, particularly with that of the perikaryon and basal pedicle. Our previous and present results strongly suggest that indole compounds, which are involved in the regulation of various neuroendocrine processes in fish, are synthetized within the CRL. Taking into account that the CRL of the pike are also photosensitive, it appears more and more likely that they are photoneuroendocrine cells involved in mediating the effects of the photoperiod on various physiological and behavioral processes.
Immunoelectron microscopic (IEM) studies on glutaraldehyde-fixed renal specimen.
Authors: Shindo N , Kobayashi E , Okada M
Published: April 2011

Immunofluorescent microscopy (IF) has become an essential tool in the routine diagnosis of renal pathology. It is thought that glomerular IF patterns represent different forms of immunologically mediated glomerular lesions. However, in order to preserve antigenicity IF is usually done on frozen, unfixed specimen by light microscopy and it is difficult to locate target macromolecules in tissue with precision. In order to establish the precise location of such macromolecules in glomeruli in relation to the ultrastructure, we undertook an immunoelectron microscopic (IEM) study on renal biopsies. Percutaneously biopsied tiny specimen was fixed in glutaraldehyde and processed with protease. Using peroxidase-labeled antisera, a direct IEM was done. With this technique, target macromolecules corresponded well not only to electron-dense deposits but also to IF patterns. In one case of membranous nephropathy, immunoglobulin (Ig) was found to be outside of the glomerular basement membrane (GBM), while IgA was seen to be inside the GBM. For this same case, a similar localization of immunoglobulins was not revealed by IF. In conclusion, IEM is a useful technique that can broaden our knowledge of pathogenic mechanisms in glomerular disease.
Histochemical localization of estrogen and progesterone receptors: evaluation of a method.
Authors: Bergqvist A , Carlström K , Ljungberg O
Published: April 2011

A histochemical method for the detection of estrogen (ER) and progesterone (PR) receptors in human endometrium, using estrogen and progesterone derivatives linked to fluorochrome-labeled bovine serum albumin (E2-BSA-fluorescein isothiocyanate (FITC) and progesterone-BSA-tetramethylrhodamine isothiocyanate (TMRITC], has been evaluated. The fluorochrome-labeled steroids were bound to the cytoplasm--preferably in glandular epithelial cells but to a lesser extent also to stromal cells. The steroid specificity of the observed binding was studied by preincubating the sections with a series of unlabled steroids and nonsteroidal, hormonally active compounds (estradiol-17 beta, diethylstilbestrol, tamoxifen, 5 alpha-dihydrotestosterone and R 1881 for ER and ORG 2058, R 5020, dexamethasone, cortisol and 5 alpha-dihydrotestosterone for PR). The inhibition studies indicated that E2-BSA-FITC and progesterone-BSA-TMRITC bind to ER and PR in human endometrium with a reasonable degree of specificity. The method was reproducible and various procedural steps were tested, showing satisfactory technical stability. The method is applicable to small tissue samples, and is a valuable complement to quantitative biochemical receptor assays, as it localizes the receptors in tissue slices.
Methods and results of testing "low acid" glycol methacrylate (GMA) for light microscopic cytochemistry.
Authors: Cole M B
Published: April 2011

Four samples of "low acid" and one sample of "technical grade" glycol methacrylate from three commercial sources were compared for their suitability in light microscopic cytochemistry. The "technical grade" was found unacceptable, but the four "low acid" products were found to be essentially the same in terms of water and ethanol miscibility, pH, polymerization time, sectioning qualities, and lack of background staining with methylene blue.
A specific ultrastructural stain for arylsulfatase A activity in human cultured fibroblasts.
Authors: Chang P L , Moudgil G
Published: April 2011

A staining reaction was developed to specifically detect arylsulfatase A activity in the presence of arylsulfatases B and C. Nitrocatechol, generated by all arylsulfatases from the substrate p-nitrocatechol sulfate, can be coupled to produce Hatchett 's brown which reacts with 3,3'-diaminobenzidine to yield an osmiophilic polymer visible under the electron microscope. The reaction was made specific for arylsulfatase A by inhibiting arylsulfatase C activity with low pH and arylsulfatase B activity with pyrophosphate. The specificity was confirmed both by electrophoretic analysis and by patient fibroblasts deficient only in arylsulfatase A activity. Under optimal conditions for preserving structural integrity and enzyme activity, enzyme reaction deposits were found mainly around vesicles. Some of these vesicles were large and heterogeneous (48-330 nm in diameter), distributed randomly within the cytoplasm, but most of the positive-reacting vesicles were uniform in size (86 +/- 18 nm in diameter) and distributed in a peripheral zone about 0.1-0.5 micron wide. These periplasmic vesicles might be partly fused with each other or with the plasma membrane. In conclusion, a specific stain for arylsulfatase A activity suitable for light and electron microscopy and the optimal conditions for structural and enzymatic preservations were developed. Although this enzyme has been considered to be lysosomal in origin, most of the activity was detected in periplasmic vesicles near the cell surface.
Immunohistochemical localization of carbamoyl-phosphate synthetase (ammonia) in adult rat liver; evidence for a heterogeneous distribution.
Authors: Gaasbeek Janzen J W , Lamers W H , Moorman A F , de Graaf A , Los J A , Charles R
Published: April 2011

Different fixation media have been compared in order to find one that preserves the histological structure of rat liver and allows unambiguous immunohistochemical detection of carbamoyl-phosphate synthetase (ammonia). Fixation of rat liver in a mixture of methanol, acetone, and water yields the most intense immunohistochemical staining. Using a specific antiserum raised against rat liver carbamoyl-phosphate synthetase, less than 1% of the enzyme protein is extractable after this fixation procedure, and the histological structure is similar to that after fixation in Bouin's fixative. Specific immunohistochemical staining is localized exclusively in the cytoplasm of the parenchymal cells; its granular distribution is in accordance with the mitochondrial localization of carbamoyl-phosphate synthetase. Immunohistochemical staining shows a heterogeneous distribution within the liver acinus. Staining is most intense around the portal venules, decreases slowly toward the hepatic venules and is, after an abrupt decrease, virtually absent in a limited area surrounding these venules. The possible significance of the heterogeneous distribution of carbamoyl-phosphate synthetase for ammonia metabolism is discussed.
The distribution of fibronectin in rat tooth and periodontal tissues: an immunofluorescence study using a monoclonal antibody.
Authors: Connor N S , Aubin J E , Melcher A H
Published: April 2011

The distribution of fibronectin (FN) in longitudinal, buccolingual sections of decalcified adult rat periodontium and teeth was studied by indirect immunofluorescence using a monoclonal antibody. FN was present in virtually all regions of the periodontium, including the gingiva, periodontal ligament, many blood vessel walls, alveolar bone, incisor and molar predentine and dentine, and molar acellular and cellular cementum. The cementum of the incisor, ameloblasts, stratum intermedium and stellate reticulum, and the connective tissue of the pulp and the surface of ondontoblasts facing the pulp in the incisor and molar were not labeled for FN. FN distribution was not always uniform either within a given connective tissue or between different connective tissues of the same organ.
Immunocytochemical localization of Mullerian inhibiting substance in the rough endoplasmic reticulum and Golgi apparatus in Sertoli cells of the neonatal calf testis using a monoclonal antibody.
Authors: Hayashi M , Shima H , Hayashi K , Trelstad R L , Donahoe P K
Published: April 2011

Mullerian Inhibiting Substance (MIS) has been localized in the Sertoli cells of the neonatal calf testis using preembedding immunoperoxidase techniques and a monoclonal antibody which almost completely blocks the biological activity of MIS. Both the peroxidase-labeled antibody method using a peroxidase-conjugated F(ab')2 fragment of IgG as a second antibody and the unlabeled antibody peroxidase-antiperoxidase (PAP) method using Fab fragments of the PAP complex were employed. With both methods, MIS was demonstrated within the cisternae of the rough endoplasmic reticulum (RER) and the Golgi apparatus. In the Golgi, MIS was concentrated in the transmost cisternae especially at their peripheral expansions. This study indicates that MIS is synthesized in the RER and transported to the Golgi apparatus, presumably for glycosidation, before secretion from Golgi derived vacuoles.
Ultrastructural immunocytochemical localization of 5-hydroxytryptamine in gastric enterochromaffin cells.
Authors: Dey R D , Hoffpauir J
Published: April 2011

Enterochromaffin (EC) cells in the gastrointestinal tract are known to contain 5-hydroxytryptamine (5HT). The probable ultrastructural localization of 5HT in the dense core vesicles ( DCVs ) of EC cells is based on the use of histochemical techniques, such as argentaffinity and the potassium dichromate reaction. In the present paper we describe an immunocytochemical method for specifically localizing 5HT in EC cells by electron microscopy. Pieces of mucosa from the pyloric region of the rabbit stomach were prepared for electron microscopy by fixation in 0.5% glutaraldehyde-picric acid-formaldehyde without osmication , and then embedded in LX-112. Thick sections (1 micron) were mounted on glass slides and processed for the fluorescence immunocytochemical localization of 5HT. Thin sections (60-90 nm) were mounted on formvar-coated slot grids and processed for the ultrastructural immunocytochemical localization of 5HT. Both the thick and thin sections were processed by an identical procedure, beginning with a 30-min incubation in anti-5HT antiserum diluted 1:1400, followed by an IgG-FITC-gold-labeled second antibody. Fluorescent EC cells were consistently observed in the thick sections of gastric mucosa. By carefully trimming and sectioning the adjacent block face, the identical EC cell could be identified by electron microscopy. A quantitative analysis revealed the number of gold particles in EC cells to be significantly greater over the cores of DCVs than over the non-core cytoplasm or over the nucleus. Absorption of the primary antiserum with 5HT abolished all labeling, while absorption with a 5HT precursor, 5-hydroxytryptophan, did not significantly reduce core labeling. Non-EC epithelial cells were not labeled. These results demonstrate that immunoreactive 5HT in EC cells is stored in the cores of DCVs .
Immunocytochemical study of proteinase F in the mouse submandibular gland.
Authors: Tanaka S , Hosoi K , Tanaka I , Kumegawa M , Ueha T
Published: April 2011

Esteroproteinases extracted from the submandibular gland (SMG) of normal male mice were fractionated by isoelectric focusing into three major peaks with isoelectric point (pI) values of 9.9 (P-esterase), 5.8 (proteinase A), and 5.6 (proteinase D). In castrated males or normal females, an additional esteroproteinase with a pI of 4.6 (proteinase F) appeared. By single radial immunodiffusion analysis using a specific anti-proteinase F serum, the proteinase F content in females or castrated males was found to be 15 times as high as that in normal males. These facts suggest that the synthesis of proteinase F is inhibited by androgens. Immunocytochemical localization of proteinase F in the SMG was examined by indirect enzyme-labeled antibody and ferritin-labeled antibody methods for light and electron microscopy, respectively. Castration of normal males caused morphological changes in granular convoluted tubular (GCT) cells, i.e., GCT cells with both several small secretory granules in their apical region and some striations at their basal region (light cells) were observed in addition to typical GCT cells. Immunoreactive proteinase F was exclusively localized in such small secretory granules of the light cells, but was only minimally present in large secretory granules of the typical GCT cells. In females, however, uniform localization of proteinase F among secretory granules of all GCT cells was observed. It is suggested that the small secretory granules in light cells are formed after castration.
Immunohistological localization of alpha 1-microglobulin in normal rat tissues.
Authors: Bouic P , Vincent C , Revillard J P
Published: April 2011

The tissue distribution of rat alpha 1-microglobulin (alpha 1-m) was studied by indirect immunofluorescence in various rat tissues using a polyvalent rabbit antiserum to the purified antigen and a monoclonal antibody (H23) to the human homologue, in parallel with a polyclonal anti-rat IgA antiserum. It was found that all tissues stained by anti-IgA were also alpha 1-m positive; these tissues included tissues of the stomach, duodenum, ileum, colon, pancreas, trachea, esophagus and jejunum. However, the observation that IgA plasma cells as well as secretory cells, while positively stained by anti-IgA, are alpha 1-m negative suggests that the association between IgA and alpha 1-m occurs at a postsecretory stage, after the IgA molecules have been transported across the epithelial cells. Additionally, hepatocytes were intensely stained by anti-alpha 1-m antibodies, indicating that the liver, as already suggested by metabolic studies on isolated guinea-pig liver explants, may be responsible for the synthesis of this protein. Among lymphoid tissues, an intense and homogeneous staining was observed in the thymus and the white pulp of the spleen. Sections of lymph nodes, however, showed differential staining; apart from a few isolated dendritic cells in the mantle region of the lymphoid follicles, the germinal centers and medullary cords showed no staining with anti-alpha 1-m antibodies. The paracortical cells, macrophages in the subcapsular sinus, and interfollicular lymphocytes showed intense cytoplasmic staining with anti-alpha 1-m antibodies. In other tissues, macrophages, monocytes, tissue histiocytes, and dendritic cells were alpha 1-m positive. Although they confirm the presence of alpha 1-m in the lymphoid tissues, as already reported in man, these results show that the protein is also present in hepatocytes and in exocrine fluids containing IgA. Since alpha 1-m, like secretory component, can bind to IgA to form stable complexes, these two heavily glycosylated proteins may have similar biologic properties.
Visual detection of granulocyte surface antigens using the avidin-biotin complex.
Authors: Henke M , Yonemoto L M , Lazar G S , Gaidulis L , Hecht T , Santos S , Blume K G
Published: April 2011

A visual test for detection of granulocyte surface markers using the avidin-biotin complex (ABC) has been developed. That this assay is highly specific, reproducible, and sensitive was determined by studying the expression of HLA antigens on granulocytes with monoclonal antibodies. Further, using granulocyte specific alloantisera, the results of the ABC test compared well to data from leukoagglutination assays and indirect immunofluorescence tests. The assay is particularly advantageous in that granulocytes can be stored, only small amounts of cells and sera are needed, and heterogeneous cell populations can easily be studied.
The electron microscopic immunohistochemistry of elastase-treated aorta and nuchal ligament of fetal and postnatal sheep.
Authors: Fukuda Y , Ferrans V J
Published: April 2011

In conjunction with the immunoperoxidase and the immunoferritin methods, antielastin antibody was used to study the localization of elastin in untreated and elastase-treated elastic fibers of the nuchal ligament and the aorta of fetal and young adult sheep. In tissues not treated with elastase, the staining reaction for antielastin antibody was localized in the outer zones of the amorphous components and along the surfaces of the microfibrils ; the central zones of the amorphous components were unreactive. After mild elastase treatment, incompletely digested amorphous components showed staining both in their central and outer zones, and some of the microfibrils became unreactive. After extensive elastase treatment, small scattered amorphous components were still found in association with bundles of microfibrils. These components were stained diffusely by the antielastin antibody method but were not detectable by staining with uranyl acetate and lead citrate or with Kajikawa 's method for elastin; elastin was not detected on the surfaces of the microfibrils by any of the methods used. These findings were interpreted as indicating that the surfaces of the microfibrils are associated with small amounts of elastin, and that evenly stained amorphous components are composed of elastin, which is loosely arranged and allows the penetration of antielastin antibody. These observations support the concept that microfibrils serve an important role as a scaffold for elastin deposition in elastogenesis. Because of their high sensitivity, immunohistochemical methods for detecting elastin are useful to study partially degraded elastic fibers.
Use of immunogold electron microscopy and monoclonal antibodies in the identification of nuclear substructures.
Authors: Clevenger C V , Epstein A L
Published: April 2011

A cytochemical technique for the ultrastructural localization of unique nuclear antigens is reported. Using a post-embedding indirect immunogold labeling procedure, nuclear antigens in electron-dense regions of the nucleus are localized with a minimum of nonspecific staining. Using this technique and indirect immunofluorescence, a panel of antinuclear monoclonal antibodies is shown to recognize preferentially cell cycle-dependent nuclear substructures. The antigenic domains recognized include specific regions in condensed chromatin, interchromatin granules, euchromatin, and chromosomes. The specificity of antigen recognition is demonstrated with qualitative and quantitative immunogold electron microscopy and immunoblot analysis. These results reveal the existence of previously undefined supramolecular organization within the nucleus and demonstrate the utility of the immunogold procedure when monoclonal antibodies are used.
The distribution of six enzymes of oxidative metabolism along the rat nephron.
Authors: Burch H B , Bross T E , Brooks C A , Cole B R , Lowry O H
Published: April 2011

Using quantitative methods, citrate synthase (CS), fumarase, beta-hydroxyacyl-coenzyme A (CoA) dehydrogenase (beta OAC), 3-keto-acid CoA transferase (KCT), malic dehydrogenase (MDH), and malic enzyme were measured in seven defined parts of the nephron and in thin limb and papilla areas dissected from freeze-dried microtome sections of rat kidney. The results not only show a wide range of activity along the nephron for each of the enzymes, but that the proportions between the enzymes vary markedly among the different parts of the nephron. This suggests the existence of major regional differences in the capacity to oxidize specific metabolites. The ratio between two citrate cycle enzymes, fumarase and CS, was 4- or 5-fold higher in proximal segments than in the glomerulus or thin limb areas. The ratio between beta OAC (an enzyme of fatty acid oxidation) and CS was 3- to 5-fold higher in the middle proximal segments than in glomeruli or thin limb and papilla areas. The key enzyme for ketone body metabolism, KCT, was essentially confined to the thick tubule segments. Malic enzyme, in contrast to the other five enzymes, was highest in the proximal straight segments. New methods, sufficiently sensitive for this histochemical study, are described for malic enzyme and 3-keto-acid CoA transferase.
Measurement of fluorescence using digital integration of video images.
Authors: Barrows G H , Sisken J E , Allegra J C , Grasch S D
Published: April 2011

The authors describe a system for the quantitation of fluorescence light output by individual cells using the signal obtained from a silicon intensifier target video camera. The video image is digitized to 4 bits (16 levels), and a 512 X 512 matrix is constructed and stored in 128K of video memory. Areas to be measured are user-specified by means of a light pen entry system. Recorded intensities are integrated under microprocessor control to provide a measure of total fluorescence in the selected areas. The distribution of light intensities per pixel over the 16-level gray scale as well as morphometric data are also obtainable. Linear response to transmission and fluorescence standards was verified. Reproducibility of the system was evaluated using fluorescent beads and glutaraldehyde-fixed chick red blood cells, which gave coefficients of variation comparable to those obtainable from other systems. Measurements of DNA per nucleus of human diploid fibroblasts using Hoechst dye 33258 yielded the two sharp peaks corresponding to the 2C and 4C values of DNA expected from such cells. We conclude that digital video measurement of fluorescence provides meaningful data and has considerable promise as a sensitive tool for the quantitation of fluorescence at the cellular level.
Light and electron microscopic localization of ACTH and pro-opiomelanocortin-derived peptides in human developmental and neoplastic cells.
Authors: Osamura R Y , Tsutsumi Y , Watanabe K
Published: April 2011

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.
Simultaneous cell surface phenotype and cell cycle analysis of lymphocytes by flow cytometry.
Authors: Noronha A , Richman D P
Published: April 2011

A method for the simultaneous measurement of cell surface components and nucleic acids (DNA and RNA) of human lymphocytes by flow cytometry has been developed, thereby providing a means of analyzing cell surface changes during the various phases of the cell cycle. Unfixed cells were coated with fluorescein-conjugated concanavalin A (F Con A) or surface antigen-specific antibody, fixed sequentially with paraformaldehyde and methanol, treated with specific nucleases, and then stained with propidium iodide. Neither portion of the procedure (cell surface staining, nucleic acid staining) interfered significantly with the other. Cell cycle phases of phytohemagglutinin-stimulated human lymphocytes as determined by this method were comparable with those identified by acridine orange staining. Cell cycle-specific blocking agents were used to additionally demonstrate the specificity of the staining procedure. Simultaneous measurement of cell cycle phase and detection of surface receptors for Con A and T lymphocyte surface determinants was performed with this method.