bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of kallikrein in the rat kidney and its anatomical relationship to renin.
Authors: Orstavvik T B , Inagami T
Published: April 2011

The anatomical relationship between kallikrein and renin in the rat kidney was investigated immunohistochemically by the peroxidase-antiperoxidase method. Kallikrein was localized to the convoluted distal tubule, starting at a point, distal to the juxtaglomerular apparatus, where the thick ascending limb of loop of Henle transformed into the convoluted distal tubule. The thick ascending limb was identified by its content of uromucoid (Tamm-Horsfall glycoprotein). Kallikrein was never observed within the juxtaglomerular apparatus itself. The kallikrein-containing tubule ended where the distal tubule submerged into the collecting duct. Renin was found in epitheloid cells of the afferent arteriole. When neighboring sections were stained for kallikrein and renin, respectively, no close anatomical relationship was observed between the kallikrein-containing and the renin-containing structures.
Distribution of mitochondrial calcium: pyroantimonate precipitation and atomic absorption spectroscopy.
Authors: Happel R D , Simson J A
Published: April 2011

Mitochondrial function is highly dependent upon the concentrations of cations within the various compartments of this organelle. Utilizing variants of the pyroantimonate precipitation method, we have undertaken to localize cations, particularly calcium, in isolated mitochondria. Precipitated cations were present in two major compartments: 1) within the intracristal space and 2) in the matrix, frequently in association with the matrix face of cristal membranes. Precipitated cations were abundant in mitochondrial samples fixed with most pyroantimonate-containing fixatives, but were usually absent in samples fixed in phosphate-buffered, pyroantimonate-containing fixative solutions. Atomic absorption spectroscopy demonstrated that Ca2+ added to mitochondrial suspensions prior to fixation was retained in the mitochondrial pellet if 0.1 M collidine was present as the fixative buffer. When the same concentration of phosphate-buffered pyroantimonate-osmium fixative was employed, however, much of the added Ca2+ was lost to the supernatant. The suborganellar localization of cations was influenced by the presence of added respiratory substrate. Incubation of mitochondria in an isoosmotic buffered sucrose medium without succinate favored localization of precipitates in the intracristal space of the outer compartment. In the presence of succinate, the localization of precipitates was shifted toward the matrix compartment. This redistribution was particularly obvious in samples to which Ca2+ had been added and was blocked by the addition of antimycin. The observation of precipitated cations in the outer mitochondrial compartment supports previous evidence for a calcium-binding protein in this compartment.
Cleavage and cross-linking of proteins with osmium (VII) reagents.
Authors: Emerman M , Behrman E J
Published: April 2011

The specific cleavage of proteins following reaction with osmium tetroxide is slowed down by tertiary amines which are known to be good ligands for osmium. Bidentate ligands are more effective than monodentate ligands. These same ligands also promote an intermolecular cross-linking reaction.
Electron microscopic demonstration of neural connections using horseradish peroxidase: a comparison of the tetramethylbenzidine procedure with seven other histochemical methods.
Authors: Carson K A , Mesulam M M
Published: April 2011

Eight methods for the electron microscopic demonstration of horseradish peroxidase (HRP) labeling have been compared in adjacent series of vibratome sections of mouse lumbar spinal cord. The tracer, a HRP-wheat germ agglutinin (WGA) conjugate, was injected into the gastrocnemius muscle complex. Following retrograde axonal transport to the lumbar motor neurons and transganglionic anterograde transport of the tracer to the dorsal horn, the HRP activity was demonstrated in eight series of adjacent sections of lumbar spinal cord using eight methods. These included procedures using tetramethylbenzidine (TMB), benzidine dihydrochloride (BDHC), o-tolidine, paraphenylenediamine-pyrocatechol (PPD-PC), and 4 methods using 3,3'-diaminobenzidine (DAB). All eight methods were able to demonstrate both retrograde labeling of motor neurons and transganglionic anterograde transport into the dorsal horn. However, there were differences in the appearance of the various reaction products under the electron microscope. In addition, differences in the distribution of the reaction products were observed by both light and electron microscopy. The largest distribution of reaction product was observed with TMB. BDHC and o-tolidine were next, followed by the DAB procedures and PPD-PC. The TMB, BDHC, and o-tolidine reaction products were all found to be suitable for electron microscopy. The TMB reaction product was electron dense and had a very distinctive crystalloid appearance that made identification of HRP-labeled neuronal profiles easy and unequivocal.
Ultrastructural localization of acidic glycoconjugates with the low iron diamine method.
Authors: Takagi M , Parmley R T , Spicer S S , Denys F R , Setser M E
Published: April 2011

The present study has applied the low iron diamine (LID) method at the ultrastructural level to demonstrate acid glycoconjugates. We have examined rat epiphyseal cartilage, human bone marrow, rat tracheal glands, and mouse sublingual glands stained with LID prior to embedment. The LID staining appeared to require postosmication for adequate visualization at the electron microscope level. Thiocarbohydrazide-silver proteinate (TCH-SP) staining of thin sections variably enhanced LID reactive sites. LID-TCH-SP stained carboxyl and sulfate groups of glycosaminoglycans in the extracellular cartilage matrix, secretory granules, and expanded Golgi saccules of chondrocytes. In human bone marrow, LID-TCH-SP variably stained the cytoplasmic granules, known to contain sulfated glycosaminoglycans, and the external surface of the plasma membrane of leukocytes. Moderately strong LID staining was observed in secretory granules in mucous tubules of rat tracheal glands, known to contain sulfated glycoproteins, and in acinar cells of mouse sublingual glands, known to contain a sialoglycoprotein. The lack of sulfated glycoconjugates in acinar cells of the mouse sublingual gland was confirmed by their failure to stain with the high iron diamine method. Thus these studies indicate that the LID and LID-TCH-SP methods are useful for the ultrastructural localization of carboxylated and sulfated glycoconjugates in extracellular and intracellular sites.
Lectin-peroxidase conjugate reactivity in normal human kidney.
Authors: Faraggiana T , Malchiodi F , Prado A , Churg J
Published: April 2011

The carbohydrate histochemistry of normal human kidney has been investigated by the use of four peroxidase-labeled lectins at the light and electron microscopic level. The results show that the lectin of Lotus tetragonolobus, specific for l-fucose, binds exclusively to the proximal convoluted tubules of the nephron. While peanut and soybean lectins, specific for D-galactose and N-acetyl-D-galactosamine, respectively, are confirmed to the collecting ducts, wheat germ lectin, specific for sialic acid and N-acetyl-D-glucosamine, stains several parenchymal structures, including the glomerular capillary wall, particularly its podocyte cell coat. Sialidase digestion reveals strong binding sites for peanut and soybean lectin in the glomeruli. At the ultrastructural level most of the binding is shown to be on the podocyte surface and within the lamina rara externa of the basement membrane. The technique represents a potentially very useful tool for the study of various pathological states in the kidney.
Peroxisomal and mitochondrial beta-oxidation in the rat kidney: distribution of fatty acyl-coenzyme A oxidase and 3-hydroxyacyl-coenzyme A dehydrogenase activities along the nephron.
Authors: Le Hir M , Dubach U C
Published: April 2011

In order to determine the localization of the peroxisomal and the mitochondrial pathways of beta-oxidation in the rat kidney, fatty acyl-coenzyme A (CoA) oxidase and 3-hydroxyacyl-CoA dehydrogenase activities were measured in glomeruli and in eight proximal and distal segments of the nephron. Structurally defined segments were dissected and analyzed with microchemical assays. The peroxisomal fatty acyl-CoA oxidase is restricted to the proximal tubule. The 3-hydroxyacyl-CoA dehydrogenase activity represents mainly the mitochondrial pathway and is similarly distributed in all cortical proximal and distal segments. It is much lower in glomeruli and collecting ducts. The distribution patterns of the two enzymes remain the same after 48 hr of starvation, although the activity of fatty acyl-CoA oxidase increases in glomeruli, proximal convolution, and collecting ducts. It is concluded that the capacity for mitochondrial beta-oxidation of fatty acids is similar in the proximal and the distal nephron. The proximal tubule possesses, additionally, a peroxisomal pathway for beta-oxidation with a capacity of the same order of magnitude as in liver cells.
Comparison of paired immunofluorescence and paired immunoenzyme staining methods based on primary antisera from the same species.
Authors: Valnes K , Brandtzaeg P
Published: April 2011

Evaluation of sequential paired staining with the unlabeled antibody peroxidase-antiperoxidase (PAP) method showed that the brown reaction product of diaminobenzidine (DAB) concealed both enzyme and antigen-antibody sites in the reagent sequence. The blue reaction product of the alternative substrate, 4-chloro-1-naphthol (CN), exerted no such blocking effect. Hence, to avoid interactions between the two PAP sequences, DAB had to be used for the first and CN for the second antigen. Complete blocking required that the DAB color reaction be of sufficient strength. When two antigens were present in the same cell, the DAB deposits inhibited staining of the second antigen unless the brown color was decreased by progressive dilution of the initial primary antibody. A mixture of brown and blue could thus reflect either concomitant staining of the two antigens or unwanted interactions between the two PAP sequences. Double staining of individual cells was, therefore, equivocal and conclusions had to be based on comparative single staining results in adjacent tissue sections. Tests carried out in several model systems showed that paired direct immunofluorescence with fluorochrome conjugates of contrasting colors (green and red) was much less time-consuming, more reliable, and of higher detection sensitivity for analyses of unbalanced mixtures of two antigens in the same cell.
Ultrastructural immunohistochemical localization of anti-invasion factor (AIF) in bovine cartilage matrix.
Authors: Wezeman F H , Childs G V
Published: April 2011

Rabbit antibodies prepared against bovine cartilage anti-invasion factor (AIF) were tested for their affinity toward antigenic sites in glutaraldehyde-fixed bovine hyaline cartilage matrix. Ultrastructural localization of the antigen-antibody complex was accomplished by the unlabeled antibody peroxidase-antiperoxidase staining technique. Unextracted and salt-extracted (1 M NaCl or 3 M GuHCl) cartilage slices were incubated with anti-AIF antibodies at a working dilution of 1:20,000. Staining occurred in unextracted matrix distributed throughout the tissue, but with regional variation in the lacunar matrix. Significantly less stain was noted in extracted tissues. The results suggest that at least certain protein components in AIF are morphologically associated with matrix complexes in aldehyde-fixed tissue.
Use of serial 1-2 micrometer paraffin sections in neuropeptide immunocytochemistry for sequential analysis of different substances contained within the same neurons.
Authors: Schrell U , Sofroniew M V
Published: April 2011

The preparation of serial 1.0, 1.5, or 2.0 micrometer paraffin sections is described. The sections are cut from paraffin blocks with surface areas of up to 5 mm x 10 mm, using glass knives and a Jung Autocut microtome. Large numbers of serial sections can easily be prepared and stained by immunohistochemistry for sequential analysis. These sections are useful for demonstrating several different substances contained within the same neurons or for detailed topographical comparison of neurons that contain different substances within the same nucleus.
Detection of immunoglobulin A by immunofluorescence in glycol methacrylate-embedded human colon.
Authors: Mozdzen J J , Keren D F
Published: April 2011

Several methods to demonstrate human immunoglobulin (Ig) A in glycol methacrylate (GMA)-embedded colon are explored. Following routine fixation in neutral buffered formalin, the tissue was dehydrated and embedded in GMA. Two-micron thick sections were heat-fixed (70 to 80 degrees C) to glass slides and then soaked in either xylene or acetone to etch the GMA. Following rehydration of the sections in serial alcohols and water, tissues were treated with phosphate buffered saline (PBS) and/or a variety of enzymes (trypsin, protease V, protease VI, pepsin, and papain) for varying periods of time. After an overnight soak in PBS the tissues were stained with fluorescein-conjugated anti-human IgA and then rinsed with PBS. This technique demonstrated excellent fluorescence of the IgA-containing plasma cells and of the discrete IgA-containing vesicles normally found in the surface epithelial cells of colon. Results were best with tissues etched with xylene and digested with 0.25 mg/ml protease V in PBS, pH 7.4, for 2 hr at 37 degrees C, followed by an overnight soak in PBS. Almost no fluorescence was seen in sections not digested with enzymes. The present method offers a simple convenient technique to perform immunofluorescence for immunoglobulin antigens on GMA-embedded tissue.
Using miniature osmotic infusion pumps to maintain tritiated thymidine exposure to cells in culture.
Authors: Neely J E , Hake D A
Published: April 2011

To provide a constant level of tracer doses of tritiated thymidine to cultured cells during continuous infusion, miniature osmotic infusion pumps were used to provide replacement thymidine. By determining the loss of isotope from the media during nonreplacement, the rate of constant infusion replacement to maintain thymidine levels was calculated. The replacement rates were similar for the three cell lines examined and allowed a standard osmotic pump infusion.
An improved method for the subcellular localization of calcium using a modification of the antimonate precipitation technique.
Authors: Slocum R D , Roux S J
Published: April 2011

A new variation of the antimonate precipitation technique, employing tannic acid in the primary aldehyde-antimonate fixative, is described for use in the subcellular localization of calcium in various tissues. Chelation studies and electron microscopic, X-ray microanalytical studies of antimonate precipitates in etiolated oat tissues indicate that calcium is the major cation localized using the present experimental protocol. Preservation of ultrastructural morphology in these tissues is greatly improved over that observed in tissues fixed with conventional antimonate-aldehyde or antimonate-osmium fixatives. The regularity and reproducibility of tissue precipitate patterns suggests that 1) penetration of the tissue by the fixative, and subsequent precipitation of calcium, is rapid and uniform and 2) ion displacement during sample preparation is negligible. Calcium appears to be immobilized efficiently in situ, with greater than 90% 45Ca retention in radiolabeled tissues prepared for electron microscopy. Quantitative aspects of calcium precipitation by antimonate in 45Ca-labeled CaCl2 solutions were examined over a wide range of calcium concentrations. Precipitation was essentially linear over the expected range of biological concentrations of calcium. Furthermore, the 3:1 antimonate to calcium ratio estimated for test tube precipitates was also established for Sb/Ca in tissue precipitates analyzed using energy dispersive x-ray microanalytical (EDX) techniques. These observations suggest that the present technique is potentially useful in the semiquantitative estimation of tissue calcium levels.
Application of the avidin-biotin-peroxidase complex (ABC) method to the light microscopic localization of pituitary hormones.
Authors: Childs G , Unabia G
Published: April 2011

The avidin-biotin-peroxidase complex (ABC) method was applied to semithin (0.5-1 micron) plastic-embedded sections of intact male rat pituitaries with the use of techniques previously developed for the peroxidase-antiperoxidase complex (PAP) method. Stains for adrenocorticotropin (ACTH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), and follicle stimulating hormone (FSH) were cleaner, more reliable, and more efficient. The ABC method allowed the use of the same high dilutions of primary antisera used with the PAP method. Incubation time was cut to a third of the time used for the PAP stain. Furthermore, if the incubation time matched that used with the PAP method, (24-48 hr), the antisera could be diluted 2- to 4-fold further. This enhanced specific staining and allowed the use of dilutions similar to those used in the radioimmunoassay. In agreement with Hsu and Raine (J Histochem Cytochem 29:1349, 1981), the ABC method produced staining after only a 1-4 hr incubation in primary antibody that was diluted optimally for the PAP complex method. The stain was weak, however, and cell counts showed that it was restricted to the fraction of the specific cell population which stored the most hormone. Our tests showed that the most convenient incubation times for optimal staining were 12-16 hr. Furthermore, the ABC method appeared to stabilize greatly the reaction for FSH and thus improved its precision and reliability.
The internal reticular apparatus of Camillo Golgi: a complex, heterogeneous organelle, enriched in acid, neutral, and alkaline phosphatases, and involved in glycosylation, secretion, membrane flow, lysosome formation, and intracellular digestion.
Authors: Goldfischer S
Published: April 2011

Its topography is one of the most characteristic features of the Golgi apparatus and the reticular nature of this organelle is evident in Golgi's first drawings, in light microscopic enzyme cytochemical preparations, and in high voltage electron micrographs of thick sections. Although individual components of the Golgi apparatus may differ in staining characteristics, morphology, contents, and enzymatic activities, they are integrated into a dynamic topographical and functional unit that is closely associated with the endoplasmic reticulum. Modulation of enzymatic activities and morphological and enzymatic heterogeneity are not surprising in an organelle that is the site of both synthetic and digestive events, including glycosylation, sulfation, formation of secretory granules and lysosomes, and the degradation of endocytized material.
Immunofluorescence localization of glycoprotein hormones in the rat anterior pituitary gland using monoclonal antibodies and polyclonal antisera.
Authors: Kofler R
Published: April 2011

A hybridoma-derived monoclonal mouse antibody against bovine luteinizing hormone (LH) cross-reacting with rat LH and conventional polyclonal rabbit antisera against rat follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH) were used for the localization of the cells producing these hormones in rat pituitary glands by the immunofluorescence double staining technique. This approach enables the simultaneous identification of two different hormones on a single tissue section. It was shown that LH and FSH are produced within the same cell type, whereas TSH is synthesized by a different cell.
Immunoelectron microscopic localization of catalase in human eosinophilic leukocytes.
Authors: Iozzo R V , MacDonald G H , Wight T N
Published: April 2011

The immunologic localization of catalase in human eosinophilic leukocytes was studied by use of rabbit anti-human erythrocyte catalase (aHEC) antiserum. Indirect immunofluorescence studies revealed intense localization of fluorescence in the cytoplasm of the eosinophils with a pattern of distribution corresponding to that of the eosinophil granules. Control samples and other nucleated blood elements remained essentially unreactive. Immunoelectron microscopy using aHEC coupled to protein A-gold complexes revealed that catalase was present exclusively within the cytoplasmic granules and equally distributed over the paracrystalline body and the homogeneous granule matrix. The presence of catalase within the paracrystalline body has not been demonstrated before with conventional cytochemical techniques. The results raise the possibility that catalase is stored in the paracrystalline body in an inactive or latent form readily available during eosinophil activation and granule release.
A simplified method of producing biologically active monomeric ferritin-insulin for use as a high resolution ultrastructural marker for occupied insulin receptors.
Authors: Smith R M , Jarett L
Published: April 2011

A new, simplified, and highly reproducible method for preparing biologically active monomeric ferritin-insulin that can be used as a high-resolution ultrastructural marker for occupied insulin receptors is described. The ferritin-insulin conjugate was stable, and contained negligible free insulin and low levels of free ferritin. The hormone conjugate behaved identically to native insulin in both binding assays and biological activity (glucose oxidation) assays. The immunological and biological activities of the insulin in the conjugate were equal. Biogel 1.5 column purification was used to yield a product monomeric in ferritin.
Applications of immunocolloids in light microscopy. Preparation of protein A-silver and protein A-gold complexes and their application for localization of single and multiple antigens in paraffin sections.
Authors: Roth J
Published: April 2011

The protein A-gold (pAg) complex, a useful reagent for electron microscopic localization of antigens in thin sections, is tested for its suitability as second step reagent in light microscopic immunohistochemistry. In addition, the preparation of colloidal silver, its complex formation with staphylococcal protein A and the application of the protein A-silver complex for antigen localization in paraffin sections is reported. The antigens were visualized in a two-step technique with specific antisera in the first incubation step and pAg or pA-silver as a general second step reagent. The pAg complex gives a red coloration of antigenic sites, whereas the pA-silver stained yellow. The contrasting color provided by the two immunocolloids allowed localization of two antigens in the same section. No color mixing occurred, showing that removal of the antibodies of the first staining sequence is unnecessary. Staining is virtually permanent with the light microscopic immunocolloid method. It is concluded that pAg and pA-silver complexes are useful as general second step reagents for the localization of a variety of antigens in paraffin sections.
Nuclear diameter in the anterior pituitary gland of the rat: effects of estrogen, bromocriptine, and haloperidol.
Authors: Jacobi J M , Lloyd H M , Meares J D
Published: April 2011

Nuclear diameter, DNA synthesis, and mitotic index in the pituitary cells of male rats and serum prolactin were measured after a period of 8 days of treatment with a dopamine agonist and an antagonist given with and without estrogen. In the absence of estrogen, the dopamine agonist, bromocriptine, diminished the mean nuclear diameter of the pituitary cells and lowered pituitary DNA synthesis, and the dopamine-blocking agent haloperidol had no effect. Estrogen increased the mean nuclear diameter, pituitary mitotic index, and DNA synthesis. Bromocriptine prevented the estrogen-induced increase in mean nuclear diameter and pituitary DNA synthesis and mitotic index were lowered. Haloperidol augmented the estrogen-induced increase in mean nuclear diameter, pituitary DNA synthesis, and mitotic index. Positive correlations were obtained between mean nuclear diameter and DNA synthesis and serum prolactin. It was concluded that nuclear diameter was influenced by both DNA synthesis and secretory activity in pituitary cells.
Ultrastructural localization of prolactin in the rat anterior pituitary glands by preembedding peroxidase-labeled antibody method: observations in normal, castrated, or estrogen-stimulated specimen.
Authors: Osamura R Y , Komatsu N , Izumi S , Yoshimura S , Watanabe K
Published: April 2011

Ultrastructural localization of prolactin (PRL) was studied immunocytochemically (preembedding peroxidase-labeled antibody method) in a variety of pituitaries, including those from 1) normal, 2) castrated, and 3) castrated and estrogen-stimulated rats. In the normal rat, PRL was observed in cisternae of the rough endoplasmic reticulum (RER), perinuclear spaces, Golgi saccules, and secretory granules. In the castrated rats, PRL cells were rather atrophic and were filled with many small PRL-positive secretory granules. RER and Golgi saccules were rather inconspicuous and were almost devoid of PRL localization. The serum PRL level was markedly lowered. With estrogen stimulation after castration, the serum PRL level was markedly elevated and PRL cells showed a pronounced increase of PRL filled cisternae in the RER. From these observations, the role of secretory granules, Golgi apparatus, and RER in hormonal secretion was defined, and it was postulated that some peptide hormones would be secreted along two alternative pathways, i.e., either 1) a long (regulated) pathway or 2) a short (accelerated) pathway, in accordance with their secretory activities, which could be altered by various stimulations such as the use of estrogen.
Histochemical distribution of certain biochemical constituents in the albumin glands of snails.
Authors: Okotore R O , Ortmann M , Karduck D , Klein P J , Uhlenbruck G
Published: April 2011

The presence in situ of the biochemical constituents galactans, proteinase-inhibitors, and erythro-agglutinin anti-A in snail's albumin gland tissues has been demonstrated by histochemical techniques using fluorescein isothiocyanate (FITC)-conjugated counterparts; namely, anti-galactan-specific lectins, different proteinases, and blood group A substance. Invariably, these constituents are localized as globular structures, predominantly within the lobules of the gland tissue. There is a good correlation between the histochemical findings and the previously reported biochemical and serological findings for these substances.
Production and characterization of antisera to synthetic thyrotropin-releasing hormone (TRH).
Authors: Chen Y F , Ramirez V D
Published: April 2011

To generate anti-thyrotropin-releasing hormone (TRH) antibodies TRH was rendered antigenic presumably by reaction of its histidine residue with bis-diazotized benzidine (BDB) coupled to bovine serum albumin (BSA). Six California white rabbits were each injected with 330 micrograms protein/ml of emulsified immunogen by the multisite intradermal immunization technique. Seven repeated injections were given at 30-day intervals using half of the original quantity of antigen. Antibodies binding 125I-TRH appeared in the serum of four of the six rabbits three months after the first injection. Five months later the sera of two rabbits bound 50% of the labeled TRH at 1:6000 final dilution. Using this antiserum a radioimmunoassay for TRH was developed in which as little as 10 pg/300 microliter unlabeled TRH can be detected. The linear range of detectable TRH was 10 to 10000 pg. No cross-reaction with various hypothalamic and pituitary hormones, neurotransmitters, neuropeptides, and BSA were detected in this immunoassay. Extracts from rat and frog hypothalami produced 125I-TRH-binding inhibition curves parallel to synthetic TRH. Samples from elutes of rat medio-basal hypothalami superfused in vitro were examined by using this antiserum. Serial dilution of superfusate showed a similar inhibitions curve. Stimulatory effect of K+ depolarization on TRH release from superfused hypothalami was inhibited in Ca2+-free ethylenediaminetetraacetic acid medium. Ouchterlony double diffusion test of the TRH antisera revealed that low levels of antibodies against albumin but not immunoglobulin G or ovalbumin were also produced. However, the immunoprecipitates could only be detected with undiluted serum. In conclusion, this antiserum generated one of the most sensitive TRH radioimmunoassay currently available in the literature. The anti-TRH serum produced by this method can be used to examine both content of TRH from several tissues as well as release from hypothalamic tissue in vitro and might be useful to trace brain TRH pathways by immunocytochemistry.
Purification and use of limulin: a sialic acid-specific lectin.
Authors: Muresan V , Iwanij V , Smith Z D , Jamieson J D
Published: April 2011

A simple and rapid method for the isolation of the sialic acid-specific lectin, Limulus polyphemus hemagglutinin (LPA), from the hemolymph of Limulus polyphemus is described. Declotted hemolymph is adsorbed to an affinity chromatographic column consisting of hog gastric mucin glycopeptides coupled to agarose and LPA is eluted in a single step with a Ca2+-free buffer, giving an apparent purification of approximately 25,000-fold. The eluted material is homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing and consists of identical subunits each of 29,000 daltons. Hemagglutination inhibition studies with horse red blood cells indicate specificity of LPA for N-acetyl- and N-glycolylneuraminic acid; binding is Ca2+ -dependent and abolished by neuraminidase treatment. LPA was covalently coupled to rhodamine and to horseradish peroxidase for use in detection of sialoglycoconjugates on cells and tissues by light and electron microscopy. Examples of the use of LPA for detection of sialoglycoconjugates in rat renal tubules and glomeruli, blood vessels in rat pancreas, and on horse red blood cells are shown. The procedures described here should prove useful as a cytochemical probe for detection of sialoglycoconjugates in a variety of systems. An accompanying article utilizes these probes for the detection of sialoglycoconjugates on the plasmalemma of adult and differentiating rat pancreatic acinar cells.
Comparative distribution of laminin, type IV collagen, and fibronectin in the rat glomerulus.
Authors: Courtoy P J , Timpl R , Farquhar M G
Published: April 2011

The distribution of laminin, type IV collagen, and fibronectin was investigated in the rat kidney cortex by immunolabeling. It was demonstrated by immunofluorescence on both unfixed cryostat sections and fixed ultracryomicrotome sections, by immunoperoxidase on fixed cryostat sections, and by immunoferritin on isolated glomerular basement membranes (GBM). This multifaceted approach provided complementary and convergent results. Distinct patterns were found for each antigen in the glomerulus and remaining kidney cortex. Laminin was localized predominantly in the GBM, where it was concentrated in the laminae rarae. Staining also occurred to a lesser extent in the mesangial matrix. Type IV collagen was evenly distributed in the lamina densa of the GBM and in the mesangial matrix. Fibronectin was most abundant in the mesangial matrix, but it could also be detected in the peripheral GBM, where it was localized in the laminae rarae. Labeling for fibronectin was particularly prominent at the endothelial-mesangial interface. The findings indicate that the three layers of the GBM differ in their composition: The lamina densa contains type IV collagen and the laminae rarae contain the two attachment proteins, fibronectin and laminin. The mesangial matrix stains for all three antigens, but it is also heterogeneous and can be subdivided into several domains--i.e., the endothelial-mesangial matrix, which is particularly rich in fibronectin, the intermesangial matrix, which contains mainly type IV collagen and fibronectin, and the GBM (where it continues over the mesangial regions), which stains most heavily for laminin.
Light microscopic histochemical detection of terminal galactose and N-acetylgalactosamine residues in rodent complex carbohydrates using a galactose oxidase--Schiff sequence and peanut lectin--horseradish peroxidase conjugate.
Authors: Schulte B A , Spicer S S
Published: April 2011

A technique was investigated for the direct visualization on paraffin sections of galactose and N-acetylgalactosamine residues terminating saccharide chains in complex carbohydrates. Sections were incubated with the enzyme galactose oxidase (GO), which oxidizes the C-6 hydroxyl of galactose or N-acetylgalactosamine (GalNAc) residues, and the resulting aldehyde was visualized by its reaction with Schiff's reagent. Submaxillary and sublingual glands, pancreas, stomach, duodenum, and ileum from mice and rats were stained with the GO-Schiff sequence and results were compared with staining by a peanut lectin-horseradish peroxidase (PL-HRP) conjugate that binds selectively to terminal galactose and preferentially to the terminal dimer beta-D-Gal-(1 leads to 3)-D-GalNAc. Three classes of reactive sites were revealed: 1) those reactive with both GO-Schiff and PL-HRP, 2) those stained with the GO-Schiff sequence but unreactive with PL-HRP, and 3) those GO-Schiff unreactive but PL-HRP positive. Based on the carbohydrate binding specificity of GO and PL, it is suggested that tissue complex carbohydrates in group one contain terminal beta-galactose residues with unmodified hydroxyls at C-2, C-4, and C-6, whereas those in group two contain terminal GalNAc residues. The structure of oligosaccharides in group 3 sites remains enigmatic.
Cytochemical demonstration of adenylate cyclase with strontium chloride in the rat pancreas.
Authors: Zajic G , Schacht J
Published: April 2011

A cytochemical procedure for the localization of adenylate cyclase with Sr2+ as the capture ion and adenylyl imidodiphosphate as the specific substrate was evaluated in the rat pancreas. Incubation medium was unaffected by the addition of 5 mM strontium ions but became turbid in the presence of lead or strontium plus 10 mM NaF. Tissues were prefixed in 2% formaldehyde/0.5% glutaraldehyde and incubated, and the cytochemical precipitate was converted to the Pb2+ salt. Enzymatic activity was demonstrated on the plasma membrane of pancreatic acinar cells and responded to stimulation by secretin. Controls frequently contained Pb2+ sequestered in mitochondria, but otherwise only a few randomly distributed grains were observed. The controls were 1) omission of substrate from the medium; 2) incubation of tissue for 1 min in complete medium; and 3) tissue previously inactivated by microwave irradiation and incubated for 30 min in complete medium including secretin.
Localization of antibody binding sites in ultrathin sections of unembedded frog retinal tissue.
Authors: Pease D C , Nir I , Clark V , Hall M
Published: April 2011

Much ultrastructural detail is retained in tissue fixed only with aldehydes and subsequently air-dried after suspension in a polyvinyl acetate emulsion. The latter provides an external support only, but permits ultrathin sectioning; thus, an exposure of intracellular contents for potential immunocytochemical reactions is achieved. Sections of unembedded frog retina so prepared have been studied with success. The tissue was incubated first with a rabbit antiserum prepared against gradient purified bovine rod outer segments. Following incubation, reacted sites were labeled with ferritin-conjugated goat anti-rabbit IgG and stained with phosphotungstic acid. Intense labeling of the rod outer segments was clearly achieved, whereas the cone outer segments were without label. Other parts of the retina, including the ellipsoid region of both rods and cones, were also without significant label. These regions provided an intrinsic control for the specificity of the antiserum and established the validity of the general technique.
Basement membrane-like matrix of teratocarcinoma-derived endodermal cells: presence of laminin and heparan sulfate in the matrix at points of attachment to cells.
Authors: Leivo I
Published: April 2011

Teratocarcinoma-derived endodermal PYS-2 cells are known to synthesize an extracellular matrix containing the basement membrane molecules laminin, type IV collagen, and heparan sulfate proteoglycan as major constituents (I. Leivo, K. Alitalo, L. Risteli, A. Vaheri, R. Timpl, J. Wartiovaara, Exp Cell Res 137:15-23, 1982). Immunoferritin techniques with specific antibodies were used in the present study to define the ultrastructural localization of the above constituents in the fibrillar network. Laminin was detected in matrix network adjacent to the basal cell membrane and in protruding matrix fibrils that connect the matrix to the cell membrane. Ruthenium red-stainable heparinase-sensitive 10- to 20-nm particles were often present at the junction of the attachment fibrils and the matrix network, or along the attachment fibrils. A corresponding distribution of ferritin label was observed for basement membrane heparan sulfate proteoglycan. Type IV collagen was found in the matrix network but not in the attachment fibrils. The results suggest that the PYS-2 cells are connected to their pericellular matrix by fibrils containing laminin associated with heparan sulfate-containing particles. These results may also have relevance for the attachment of epithelial cells to basement membranes.
Histochemical properties of cartilage proteoglycans.
Authors: Ippolito E , Pedrini V A , Pedrini-Mille A
Published: April 2011

Proteoglycan interaction with alcian blue at different concentrations of magnesium chloride was studied both in vitro and in histological sections of paraffin-embedded tissues. Our experiments indicate that a) proteoglycans with different contents of chondroitin sulfate and keratan sulfate, prepared under nondegradative conditions, are not distinguishable on the basis of the critical electrolyte concentrations at which staining is abolished; b) the state of aggregation of proteoglycans only very slightly affects the alcian blue affinity of the macromolecules at different concentrations of magnesium chloride; c) the interaction of proteoglycans with other components of the connective tissue matrix is an important factor in determining the strength of binding of alcian blue to the polyanionic macromolecules in histological sections. These factors should be considered in interpreting histochemical data obtained by staining tissue sections with alcian blue at different concentrations of magnesium chloride. Proteoglycans, like glycosaminoglycans, are only weakly periodic acid-Schiff-positive.
Segmentation of cell nuclei in tissue section analysis.
Authors: Zinser G , Komitowski D
Published: April 2011

Image segmentation is a critical step in digital picture analysis, especially for that of tissue sections. As the morphology of the cell nuclei provides important biological information, their segmentation is of particular interest. The known segmentation methods are not adequate for segmenting cell nuclei of tissue sections; the reason for this lies in the optical properties of their images. We have developed new gradient methods of segmentation of previously presegmented images by taking these properties into account and by using the approximately circular shape of the cell nuclei as a priori information. In our first technique, the segment method, the images of the nuclei are divided into eight segments, special gradient filters being defined for each segment. This has enabled us to improve the gradient image. After searching for local maxima, the contours of nuclei can be found. In the second method, the method of transformation into the polar coordinate system (PCS), the a priori information serves to define a circular direction field for gradient computation and contour finding. In contrast with the first method, which offers a rapid, general idea about the nuclear shape, the PCS method permits precise segmentation and morphological analysis of the cell nuclei.
Characterization of basal lysosomes in exocrine acinar cells.
Authors: Oliver C
Published: April 2011

Exocrine acinar cells possess a unique system of basally located lysosomes. Cytochemically, these lysosomes do not contain acid phosphatase, but react positively for trimetaphosphatase (C Oliver: J Histochem Cytochem 28:78, 1980). The present study extends the morphological and cytochemical characterization of these lysosomes in pancreatic, parotid, and exorbital lacrimal acinar cells from Sprague-Dawley rats and National Institutes of Health Swiss mice. The basal lysosomes are highly pleomoric in nature, and frequently appear as a system of anastomosing tubules of varying width. The lysosomes have a close morphological relationship with both the rough endoplasmic reticulum and mitochondria. In addition to trimetaphosphatase activity, the lysosomes are reactive for aryl sulfatase B, thiolacetic acid esterase, and cholinesterase. Since the cholinesterase activity could not be inhibited by specific inhibitors, this activity is most likely due to the presence of nonspecific esterases. The results of this study confirm the lysosomal nature of the basal lysosomes and underscore the necessity of using multiple enzyme activities to identify and characterize lysosomes.
Characterization of tumor cell membrane serine proteases by polyacrylamide gel electrophoresis.
Authors: Distefano J F , Beck G , Zucker S
Published: April 2011

Studies in our laboratory have indicated that tumor cell membrane-bound proteases are responsible for the ability of tumor cells to lyse normal cells in vitro. In order to evaluate the tumor cell membrane enzymes, a purified tumor cell membrane preparation was prepared and the nonionic detergent Triton X-100 was used to extract active enzymes from the cell membranes. The solubilized membrane enzymes were then studied by Triton X-100 polyacrylamide gel electrophoresis under non-denaturing conditions. Using this technique the tumor cell membranes were shown to contain esterproteases that reacted with the substrates alpha-naphthyl acetate and naphthol-AS-aminocaproate. These esterproteases were inhibited by diisopropyl fluorphosphate and tosyl lysine chloromethyl ketone but not by tosylamide phenylethyl chloromethyl ketone, soybean trypsin inhibitor p-chloromercuribenzene sulfonic acid; N-ethylmaleimide choline iodide, alpha-1-anti-trypsin. NaF, epsilon-aminocaproic acid, ethylenediamine tetraacetic acid, or eserine. SBTI affinity chromatography of the tumor cell membrane extract revealed that some of the serine esterproteases bound to the SBTI column. The proteolytic activity of the tumor cell membrane extract and a fraction eluted from the SBTI affinity column was demonstrated using casein. We conclude that the tumor cell membranes contain previously undescribed serine proteases that are identifiable by their esterase activity and inhibitor profiles in polyacrylamide gels.
Antibodies to hepatocyte membrane antigens in chronic liver disease: detection by immunofluorescence after Bouin's fixation.
Authors: Lee W M , Shelton L L , Galbraith R M
Published: April 2011

Using a modified indirect immunofluorescent (IF) technique in which cryostat tissue sections were fixed in Bouin's solution for ten minutes prior to reaction with sera under test, we have looked for antibodies to the hepatocyte membrane (HMA) in the sera of patients with chronic active hepatitis (CAH) and primary biliary cirrhosis (PBC). Samples were tested initially in parallel on unfixed and Bouin's-fixed rat composite blocks (kidney, stomach, liver) at a titer of 1:100 and those found to be positive were diluted further and reexamined. Conventional unfixed sections of rat composite block showed no liver membrane immunofluorescence although antinuclear (ANA), mitochondrial (AMA), and smooth muscle antibodies (SMA) were detected as anticipated. By contrast, prior Bouin's fixation abolished most of the fluorescence due to ANA, AMA and SMA but resulted in brilliant fluorescence of rat hepatocyte membranes in eleven of twelve patients with CAH (93%) and all ten patients with PBC. Only one of 22 normals (5%), one of 20 with collagen-vascular diseases (5%), and one of seven with nonimmunologic liver disease (14%) were positive for this hepatocyte membrane antibody. Bouin's fixation prior to IF is a simple technique which appears to alter the hepatocyte membrane so that HMA become detectable. The strong association of HMA with CAH and PBC suggests that this test might be of value and may contribute towards a further understanding of the pathogenesis of these conditions.
Immunolocalization of the protease kallikrein in the colon.
Authors: Schachter M , Peret M W , Billing A G , Wheeler G D
Published: April 2011

Colon kallikrein was localized in the goblet cells of cat and man by a variety of immunocytochemical techniques. No evidence of this enzyme was found in other sites in this organ. The possible physiological significance of kallikrein in the gastrointestinal tract and of the many related serine proteases is discussed.
Immunohistochemical localization of glandular kallikrein in the endocrine and exocrine human pancreas.
Authors: Pinkus G S , Maier M , Seldin D C , ole-MoiYoi O K , Austen K F , Spragg J
Published: April 2011

Fab fragments from two new monospecific anti-human tissue kallikrein sera were examined for their capacity to inhibit the functional activities of purified human urinary kallikrein and purified human pancreatic kallikrein. Fragments from a new anti-urinary kallikrein serum and from an anti-pancreatic kallikrein serum yielded mixed inhibition of kinin-generating activity and minimal inhibition of esterolytic activity. In contrast to the previously described "active site directed" anti-urinary kallikrein, these new antisera demonstrated little specificity for epitopes near the enzymatic site of urinary or pancreatic kallikrein. When used to localize kallikrein antigen in human pancreas obtained at surgery, IgG fractions of the new anti-kallikrein sera yielded moderate acinar and ductal staining in the absence of pretreatment of the tissue with trypsin or pronase. Short incubation with 0.125 mg/ml of either enzyme permitted the discrete localization of islet beta cell kallikrein antigen, while increased pronase concentrations decreased kallikrein antigen in both islets and exocrine tissue and led to islet destruction. Both antibody specificity and tissue preparation influence kallikrein localization in human pancreas.
Pattern and rate of disappearance of gamma-glutamyl transpeptidase activity in fetal and neonatal rat liver.
Authors: Iannaccone P M , Koizumi J
Published: April 2011

Gamma-glutamyl transpeptidase (gamma-GTP), an amino acid transport enzyme, has been demonstrated in a number of fetal and adult tissues of rodent and man. While the re-expression of the enzyme has been described in epithelia following carcinogen treatment or aging, little is known of the mechanism of its disappearance in some neonatal tissues. A description is presented of the rate and pattern of loss of histologically demonstrable gamma-GTP activity from fetal and neonatal liver of the rat. The number of hepatocytes with histologically demonstrable gamma-GTP activity declines rapidly from the 18th day of gestation. By the 6th day postpartum the activity is demonstrable in clusters of hepatocytes. By the 7th day postpartum there are essentially no hepatocytes with demonstrable activity, although the enzyme remains expressed in bile duct epithelium.
Detection of a light-sensitive pool of cGMP in goldfish cone color receptors by immunocytochemistry.
Authors: Ortez R A , Tamayo A , Johnson C , Sperling H G
Published: April 2011

A procedure for the immunocytochemical localization of cGMP in the goldfish retina is described. The procedure is then used to evaluate the presence of cGMP in cone color receptors in retinas from dark-adapted and light stimulate fish. The results show that cone color receptors in dark-adapted retinas contain large amounts of immunocytochemically defined cGMP in the ellipsoid region, which are greatly reduced after light exposure.