bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of factor VIII/von Willebrand factor antigen by immunoelectron microscopy in human endothelial cells using Fab fragments coupled to peroxidase.
Authors: Jeanneau C , Sultan Y
Published: April 2011

Factor VIII/von Willebrand factor antigen (VIII R:Ag) has been localized in human endothelial cells by an immunoperoxidase method and electron microscopy. Specific antibodies against VIII R:Ag were isolated from monospecific rabbit antisera by an immunoadsorbant. Fab fragments were prepared and coupled to peroxidase. Native human endothelial cells from umbilical cord veins were incubated with the Fab conjugates. Peroxidase labeling was seen on the plasma membrane, in vesicles, and in the Golgi apparatus. In order to eliminate nonspecific antibody internalization, the same procedure was reproduced on fixed endothelial cells and similar localization was observed. Staining was only found when specific VIII R:Ag Fab conjugates were used. All the controls were devoid of labeling, thus the peroxidase reaction product localized in the Golgi cisternae is specific for VIII R:Ag and suggests VIII R:Ag synthesis in endothelial cells.
Regional differences in lectin binding to colonic epithelium by fluorescent and electron microscopy.
Authors: Gorelick F S , Sarras M P , Jamieson J D
Published: April 2011

The distribution of glycosubstances in colonic epithelium using lectins that were rhodaminated for fluorescent microscopy or coupled to colloidal gold for electron microscopy has been studied. Findings, based on lectin binding patterns, indicate that the right, transverse, and left colon of the guinea pig differ in their content of glycoconjugates within goblet cells and along the brush border. Local variations of labeling were also observed within goblet cells between the upper and lower portions of crypts. Throughout the colon, corresponding to a region of supranuclear fluorescence, Ricin II-colloidal gold labeled the Golgi complex of both enterocytes and goblet cells. Ricin II-colloidal gold also labeled small (congruent to 800 A) vesicles in the apical portion of enterocytes in all colonic segments. Microvilli were labeled by Ricin II-colloidal gold in the right and transverse colon, a finding that correlated with the observed adhesion of bacteria in those segments.
A monoclonal antibody to vasopressin: preparation, characterization, and application in immunocytochemistry.
Authors: Hou-Yu A , Ehrlich P H , Valiquette G , Engelhardt D L , Sawyer W H , Nilaver G , Zimmerman E A
Published: April 2011

The hypothalamo-neurohypophysial system, containing the hormones oxytocin (OT) and vasopressin (VP) and their associated carrier proteins, the neurophysins (NPS), has been the subject of extensive investigation for more than 40 years. This system has been reinvestigated during the last decade by application of immunocytochemical methods employing the rabbit antisera to the hormones and NPS. In this study we describe the preparation and characterization of a monoclonal antibody to VP and its application in immunohistochemistry. The antibody did not cross-react with OT or arginine vasotocin (AVT). Its antigenic determinants as characterized by absorption with various VP analogs included two aromatic amino acids: Phe in position 3, and to a lesser extent Tyr in 2. Tissue fixation with formaldehyde resulted in inadequate immunostaining as compared to glutaraldehyde, most likely due to interference with the aromatic amino acid determinants by the former fixative.
Synthesis and immunocytochemical localization of alpha 2u globulin in the duct cells of the rat submaxillary gland.
Authors: Antakly T , Laperche Y , Feigelson P
Published: April 2011

Alpha 2u globulin, a protein of unknown function so far believed to be synthesized exclusively in the male liver under multihormonal control, is now shown to be localized by immunocytochemistry in the granular convoluted tubules of the adult male submaxillary gland. In addition, using Northern blot analysis, we have shown specific alpha 2u globulin mRNA sequences in the RNA extracted from the submaxillary gland. Thus, it is evident that the protein is being synthesized therein. Alpha 2u globulin was also detected in the submaxillary gland duct cells of adult female and immature animals of both sexes, all of which are known not to synthesize alpha 2u globulin in their livers. The present data have established that alpha 2u globulin is synthesized in the rat submaxillary gland and indicate that the control of alpha 2u globulin gene expression in the rat liver and in the submaxillary gland is different.
Carbonic anhydrase activity in skeletal muscle fiber types, axons, spindles, and capillaries of rat soleus and extensor digitorum longus muscles.
Authors: Riley D A , Ellis S , Bain J
Published: April 2011

Carbonic anhydrase (CA) activities were studied in soluble extracts and cryostat sections of skeletal muscles from prepubertal and postpubertal rats. Acetazolamide inhibition was utilized to distinguish between activities of the acetazolamide-sensitive (CA I and II) and acetazolamide-resistant (CA III) forms of the enzyme. The inhibition studies indicated that fast-twitch oxidative-glycolytic muscle fibers contained both the sensitive and resistant forms of CA. Acetazolamide-sensitive activity was localized within muscle fibers, axons, myelin, and capillaries. Axoplasmic staining was restricted to subpopulations of myelinated axons in both the dorsal and ventral roots. Soleus muscles exhibited significantly greater activity of CA III than extensor digitorum longus muscles at all ages examined. CA III was richest in slow-twitch oxidative and intrafusal fibers. During puberty, soleus muscle fibers matured and converted from fast-twitch oxidative-glycolytic to slow-twitch oxidative fibers. There was a shift from the sensitive to the resistant form of CA; CA III activity increased about sevenfold. This activity peaked earlier in the muscles of female rats than male rats. These results demonstrated a complex distribution of CA isozymes in the neuromuscular system and pointed out that isozyme content depends on both the type of muscle and the age and sex of the animal.
Unconventional application of standard light and electron immunocytochemical analysis to aldehyde-fixed, araldite-embedded tissues.
Authors: Hogan D L , Smith G H
Published: April 2011

A simple procedure for the immunocytochemical analysis of glutaraldehyde/formaldehyde-fixed, Araldite- or Epon-embedded tissues by either light or electron microscopy is presented. Retention of immunoreactive antigen in deplasticized sections was achieved by use of a low concentration of glutaraldehyde in the fixative in combination with a seldom-used plastic solvent. This protocol produced good ultrastructural preservation in tissues and large, high-quality, 2-micrometers thick, plastic-free sections. These semithin sections provided a level of structural and antigenic preservation, image resolution, and labeling intensity that surpassed all other conventional sectioning methods used for immunocytochemistry. The capacity to use a single tissue sample in studies designed for light and electron immunocytochemistry, in conjunction with existing autoradiographic and cytochemical techniques, makes this a very desirable method for routine tissue preparation in research and clinical applications.
Postembedding immunocytochemical localization of paramyxovirus antigens by light and electron microscopy.
Authors: Schwendemann G , Wolinsky J S , Hatzidimitriou G , Merz D C , Waxham M N
Published: April 2011

A postembedding method is described to localize antigens specific for various paramyxoviruses in sections of cells and tissues that have been fixed and embedded in epoxy resins for conventional electron microscopy. Viral antigens were localized in CV-1 cell cultures infected with simian virus 5 (SV5), brains of suckling hamsters inoculated with either neuroadapted mumps virus or hamster-adapted measles virus, and brains of adult mice infected with Sendai (parainfluenza I) virus. Both 1-micrometer-thick and thin (gold) tissue sections were etched with alcoholic sodium hydroxide-solution and then treated following either the unlabeled antibody peroxidase-antiperoxidase or the biotinylated protein A:avidin peroxidase procedure. Primary reagents included immunoglobulin isolated from hyperimmune rabbit sera with specificity to the major viral components of SV5 or SV5 hemagglutinin-neuraminidase, to whole mumps virus or mumps virus nucleocapsids, and to whole Sendai virus. Crude rabbit anti-Sendai virus antiserum and whole human subacute sclerosing panencephalitis (SSPE) sera were used in parallel. The results indicate that tissues processed for conventional evaluation by electron microscopy may be suitable, within limits, for postembedding immunocytochemical staining of paramyxovirus antigens.
Use of Hoechst 33342 for cell selection from multicell systems.
Authors: Durand R E
Published: April 2011

Hoechst 33342 staining of multicell spheroids, three-dimensional cell clusters grown in vitro, results in a marked gradient of cellular fluorescent intensities inward from the spheroid periphery. The penetration of the dye is concentration and time dependent, so staining can be coupled with fluorescence activated cell sorting techniques to allow disaggregated single cells to be sorted or selected according to their degree of staining and therefore their depth within the spheroid. We have found the staining procedure to be highly reproducible, and to result in minimal toxicity even to the more brightly staining external cells. Comparison of this technique with others for cell selection suggests that increased resolution is available with the Hoechst technique.
The role of paraldehyde in the rapid preparation of aldehyde fuchsin.
Authors: Nettleton G S
Published: April 2011

Preparation of aldehyde fuchsin normally requires ripening for 3 to 5 days. By using a 5-fold excess of paraldehyde a fully potent aldehyde fuchsin can be prepared in 24 hr at room temperature. Aldehyde fuchsin prepared by both normal and accelerated ripening afforded comparable results, including selective staining of unoxidized pancreatic B cells. Dried aldehyde fuchsin prepared form pararosaniline and reconstituted in acid alcohol has spectrophotometric properties different form the ripened strain. Reconstituted aldehyde fuchsin stains unoxidized B cells adequately only if staining time is extended. Excess paraldehyde added to reconstituted aldehyde fuchsin retards decomposition but does not produce a normal stain by spectrophotometric standards. Warming of aldehyde fuchsin solutions to accelerate ripening has been shown to produce deleterious effects and should be avoided.
Immunocytochemical and autoradiographic demonstration of prolactin binding to human breast cancer cells in tissue culture.
Authors: Paterson J A , Salih H , Shiu R P
Published: April 2011

Specific binding of human prolactin to human breast cancer cells in culture was demonstrated for the first time by immunocytochemistry ad autoradiography. Using the former technique, it was found that prolactin binding to the cell surface was heterogeneous: some cells showing intense to moderate peroxidase reaction product and some cells showing absence of reaction product. After the cells were incubated with 125I-labeled prolactin and then embedded in plastic, autoradiography of sections revealed both surface-localized and intracellular silver grains, the latter suggesting internalization of prolactin. These two cytological techniques are useful for visualizing prolactin binding to human breast cancer cells and are applicable to studies on prolactin binding in human breast cancer.
Immunohistochemical identification of mast cells in paraffin- and epon-embedded tissues using platelet factor 41.
Authors: Shaw S T , Roche P C , Schmer G , Macaulay L K
Published: April 2011

An immunoperoxidase method has been developed for staining heparin in the granules of mast cells. The method employs human platelet factor 4 (or anti-heparin) and a rabbit antiserum to this polypeptide. Platelet factor 4 binds to mast cell heparin and provides the basis for immunoperoxidase staining using the rabbit antiserum. Preliminary studies of mast cells in various tissues indicate that the stain is quite specific for the content of mast cell granules, presumably heparin and possibly other glycosaminoglycans.
Ultrastructural localization of concanavalin A and wheat germ agglutinin binding sites in adult and embryonic mouse myocardium.
Authors: Gros D , Bruce B , Challice C E , Schrevel J
Published: April 2011

The lectins, concanavalin A (Con A) and wheat germ agglutinin (WGA), have been used to localize with precision glycosyl residues in adult and embryonic mouse myocardium. They were detected by means of an affinity method using peroxidase and chitobiosylperoxidase, respectively, which then were revealed with 3,3'-diaminobenzidine and H2O2. Exhaustive controls have shown that the binding of Con A and WGA is reversible when experiments are performed with adult specimens (tissue blocks or ultrathin sections of glycol methacrylate-embedded material) or with isolated embryonic cells. Experiments carried out with tissue blocks from embryonic hearts have shown peroxidase binding. This finding is discussed on the basis of the presence of the endogenous lectin-like components in embryonic hearts. Results show that the surface of adult and embryonic myocardial cells specifically bind both Con A and WGA, thus indicating the presence of glycosyl residues similar to alpha-methyl-D-mannoside and N-acetyl-D-glucosamine. In adult heart the transverse tubular system was also labeled. The absence of Con A and WGA receptor sites in the gap junction regions was demonstrated by means of an electron microscope postembedding staining method.
Cartilage calcification: an ultrastructural, histochemical, and analytical x-ray microprobe study of the zone of calcification in the normal avian epiphyseal growth plate.
Authors: Davis W L , Jones R G , Knight J P , Hagler H K
Published: April 2011

Sections from the zone of calcification of ruthenium red-fixed normal avian epiphyseal growth plates were analyzed by various morphological (histochemical) and analytical techniques. Calcium and phosphorus were identified in the chondrocyte pericellular rim, the uncalcified extracellular (territorial) matrix, and in both the peripheral and central aspects of the calcified accumulations within extracellular matrix. Cartilage proteoglycan, as determined by ruthenium binding, positive staining with acidic phosphotungstic acid, and the X-ray spectroscopic detection of sulfur, was identified in the same four zones. Thus, it appears that proteoglycans, in some form, are indeed retained at sites of biological calcification. Additionally, these macromolecules, synthesized in chondrocytes, may be involved in extracellular calcium translocation.
Cell types associated with fibronectin in long-term mouse bone marrow cultures.
Authors: Reincke U , Hsieh P , Mauch P , Hellman S , Chen L B
Published: April 2011

The formation of fibronectin matrix was studied in long-term mouse bone marrow cultures. Stromal and hematopoietic cells were observed in situ under phase contrast optics and quantified according to their staining characteristics on smear preparations. Surface fibronectin was demonstrated by indirect immunofluorescence. While only stromal and no hematopoietic cells participated, various stromal cell types differed in their expression of cell surface fibronectin: Reticulum cells contributed the major portion of fibronectin matrix. Elongated, meshwork-forming histiocytes expressed some surface fibronectin, while the flattened, macrophagic histiocytes remained fibronectin negative. These findings were recapitulated during regeneration of scrape wounds in the adherent layers. Isolated fibronectin matrix did not support hematopoietic cell adherence or maintenance, although it had marked effects on stromal cells.
Artifacts associated with mithramycin fluorescence in the clinical detection and quantitation of aneuploidy by flow cytometry.
Authors: Cunningham R E , Skramstad K S , Newburger A E , Shackney S E
Published: April 2011

Ethanol-fixed cells stored at 4 degrees C exhibit fixation time-dependent hyperchromatism in comparison with freshly fixed cells when stained with mithramycin and examined by flow cytometry. This hyperchromatism has been found to be temperature-dependent, developing fully within 72 hr at room temperature, and within 2 hr at 37 degrees C. Cells from normal donors that are stained with mithramycin exhibit spurious aneuploid peaks. These spurious aneuploid peaks can be eliminated by incubating ethanol-fixed cells at 37 degrees C for 2 hr prior to staining; true aneuploidy is not affected by this procedure. In rare instances, cytoplasmic fluorescence can be observed in mithramycin-stained cells. In addition, unexplained hypochromatism and hyperchromatism can be observed in some clinical samples, particularly in human melanoma. The effects of these unexplained staining artifacts can be minimized or eliminated by adopting strict criteria for the clinical detection of aneuploidy by flow cytometry.
Immunohistochemical localization of carbonic anhydrase isoenzyme C in the central and peripheral nervous system of the mouse.
Authors: Kumpulainen T , Korhonen L K
Published: April 2011

The regional and cellular distribution of the high activity carbonic anhydrase isoenzyme (CA C or CA II) in the mouse nervous system was investigated by an indirect immunoperoxidase (peroxidase-antiperoxidase) method using cross-reactive antibodies prepared against human CA C. In the mature brain an overall strong CA C specific reactivity was revealed in the heavily myelinated nerve tracts, the main immunostaining originating from small, intensively reacting cells interpreted as oligodendrocytes and from the myelin sheaths. An obvious straining was also revealed in the choroid plexus cells, especially in their free borders, and in the erythrocytes of the blood vessels, while the glial cells of the retina similarly exhibited a strong reaction. In the developing brain, CA C was absent shortly after birth, but achieved almost the mature pattern of distribution within about 3 weeks. In the spinal cord most of the positive cells were found in the grey matter, their processes projecting towards the white matter. No reaction was obtained in the sciatic nerve fibers or in the neuronal or satellite cells of the coeliac ganglion.
Identification of carbonic anhydrase in chick growth-plate cartilage.
Authors: Gay C V , Anderson R E , Schraer H , Howell D S
Published: April 2011

Carbonic anhydrase has been localized by immunocytochemistry in the cells and territorial matrix of the hypertrophic and calcifying zones of chick growth-plate cartilage. Adjacent epiphyseal and articular cartilage were not stained. By biochemical assay the activity levels were 61.3, 1.8, 34.7, and 703.3 units/g wet weight for growth plate, epiphyseal/articular cartilage, spongiosa, and blood, respectively. The role of the enzyme in growth plate is discussed.
An ultrastructural and enzyme cytochemical study of the response of the neutrophil series to a local inflammatory stimulus in the rat.
Authors: Williams D M , Gillett R , Linder J E
Published: April 2011

Changes in neutrophil precursor populations in the bone marrow and their alkaline phosphatase reactivity following an inflammatory stimulus were studied in the rat using an osmiophilic method. Seven groups each of 3 Sprague-Dawley rats received subcutaneous injections of turpentine, and femoral marrow was examined at intervals up to 72 hr. Depletion of mature neutrophils resulted in an increased first in the myeloblast-promyelocyte compartment and at 48-72 hr in the myelocyte-metamyelocyte population. By 72 hr replenishment of the mature neutrophil marrow population had begun. Within 6 hr marked acceleration of cytoplasmic maturation was evident, together with accelerated synthetic activity, manifested by marked dilatation of the rough endoplasmic reticulum and an enlarged Golgi. Together with these changes there was an increase in the number of alkaline phosphatase reactive cells, which was evident first in the myeloblast-promyelocyte population at 2 hr. An absolute increase in the amount of enzyme reaction product associated with individual cells was also seen.
The kinetics and homogeneity of endocytosis of a receptor-bound ligand in a heterogeneous cell population studied by flow cytofluorometry.
Authors: Metezeau P , Djavadi-Ohaniance L , Goldberg M E
Published: April 2011

A method is described to study quantitatively and rapidly the kinetics of endocytosis of a receptor-bound ligand by cells of various subclasses within a heterogeneous cell population. The time course of the internalization of the bound ligand is followed by labeling, with a fluorescent antibody, the ligand molecules exposed on the cell surface at various times of the endocytosis process, and by measuring with a flow cytofluorometer the fluorescence of a large number of individual cell within the total cell population. A convenient mathematical treatment of the data is proposed for analyzing the experimental results. This method is applied to the kinetic study of the endocytosis of rabbit antibodies (anti-mouse immunoglobulin) by B-lymphocytes within a mouse spleen cell suspension.
A method for quantitative extraction of enzymes and metabolites from tissue samples in the milligram range.
Authors: Pette D , Reichmann H
Published: April 2011

A method is described for quantitative extraction of soluble enzyme activities from tissue samples in the milligram range. Mechanical disintegration of the frozen tissue is performed in a micromortar from which the powdered tissue can be quantitatively transferred for extraction in small volumes within a microcentrifuge tube.
Sponge glycoconjugates: immunological properties and localization by fluorescent antibodies and lectins.
Authors: Junqua S , Garrone R
Published: April 2011

By immunodiffusion and immunoelectrophoresis, glycoconjugates previously isolated from a sponge, Spongia officinalis, and fractionated on lectins, showed identical immunological behaviour which was species specific. By fluorescent antiserum and lectins, these glycoconjugates were located on sections of two sponges, S. officinalis and Chondrosia reniformis. A strong pericellular staining occurred together with a diffused labeling.
In vivo effects of prostaglandin E2 and arachidonic acid on phagocytosis of fluorescent methacrylate microbeads by rat peritoneal macrophages.
Authors: Fernández-Repollet E , Mittler R S , Tiffany S , Schwartz A
Published: April 2011

Several studies have suggested that prostaglandin E2 (PGE2) might influence the phagocytic activity of macrophage cells. The present study was designed to examine the in vivo effects of PGE2, the prostaglandin synthesis inhibitor meclofenamate, the prostaglandin precursor arachidonic acid, and the biologically inactive fatty acid 11,14,17-eicosatrienoic acid on phagocytosis by peritoneal macrophage cells in the rat. Following 3 days of treatment with either agent, fluorescent methacrylate microbeads were injected intraperitoneally into all rats. Peritoneal exudates were harvested after administration of the microbeads and the percent phagocytosis determined in macrophage cells using a fluorescence-activated cell sorter (FACS II). The administration of PGE2 was associated with a significant decrease in the percentage of peritoneal macrophages ingesting the fluorescent methacrylate microbeads. In contrast, treatment with arachidonic acid or 11,14,17-eicosatrienoic acid significantly enhanced the percentage of phagocytic macrophage cells. A significant increase in the number of macrophages undergoing phagocytosis of the methacrylate microbeads was also observed in rats treated with meclofenamate. This later observation, taken together with the inhibitory effect induced by PGE2 on macrophage phagocytosis, points to a potential modulator role of PGE2 on the phagocytic activity of macrophages. These data also suggest that arachidonic acid might influence macrophage phagocytosis by a mechanism independent of PGE2.
Immunohistochemical localization of fibronectin in human and rat uterine cervices.
Authors: Leppi T J , Repesh L A , Furcht L T , Bartzen P J , Holt G E
Published: April 2011

Paraffin sections of 95% ethanol-fixed uterine cervices from adult, cycling rats and from human patients undergoing elective hysterectomy were immunostained by the peroxidase-antiperoxidase technique after exposure to antisera against fibronectin. Light microscopic study of uterine cervical stroma from both species showed an irregular distribution of fibronectin between compactly arranged collagenous fibers and a more uniform deposition around scattered bundles of smooth muscle fibers. In loosely arranged areas of stromal collagen, fibronectin was found in a filamentous, occasionally punctate, distribution. There was moderate to heavy staining with immunoperoxidase for fibronectin in the regions of the basement membrane underlying cervical epithelia and in the connective tissue stroma immediately subjacent to the basement membrane. Fibronectin was also present in the walls of most blood vessels and in the connective tissue investments of peripheral nerve fascicles. These findings suggest that fibronectin may be another component of extracellular matrix in the uterine cervical wall of non-gravid human subjects and rats in addition to collagen and glycosaminoglycans.
Microanalysis of the reaction product in Karnovsky and Roots histochemical localization of acetylcholinesterase.
Authors: Tewari J P , Sehgal S S , Malhotra S K
Published: April 2011

X-ray energy dispersive microanalysis of the reaction product in Karnovsky and Roots histochemical localization of acetylcholinesterase indicated the presence of sulfur, iodine, copper, and iron. The reaction was run in vitro using purified acetylcholinesterase from the electric eel to confirm our previous results on similarly treated neuromuscular junction in situ.
Long-term storage and regular repeated use of diluted antisera in glass staining jars for increased sensitivity, reproducibility, and convenience of single- and two-color light microscopic immunocytochemistry.
Authors: Sofroniew M V , Schrell U
Published: April 2011

A procedure is described for the dilution and storage of antisera in glass staining jars into which whole slides are immersed for incubation during light microscopic neuropeptide immunocytochemistry. Diluted antisera, stored at 4 degrees C and continuously reused, were found to be stable for long periods of time (to date over 3 years), and consistently yielded high quality staining in both single- and two-color immunoperoxidase staining. We found this procedure to be more convenient than conventional incubation procedures, allowing the more rapid processing of large numbers of slides and reducing the loss of slides due to technical errors. The consistency and reproducibility of day to day staining were also improved. The immersion of whole slides into the antisera permitted the use of long incubation times (up to 7 days) without the sections drying out, which in many cases substantially enhanced the sensitivity of the staining obtained. A procedure for two-color immunoperoxidase staining is described using diaminobenzidine for a brown color and alpha-naphthol/pyronin for a red/purple color. We found the alpha-naphthol/pyronin reaction superior to the more commonly used 4-chlornaphthol reaction as a second color. The two-color staining was found useful not only for demonstrating nerve cell bodies stained different colors, but also for staining nerve terminals one color that are around and contacting nerve cell bodies stained another color.
Immunohistochemical localization of human immunoglobulins and lysozyme in epoxy-embedded lymph nodes: effect of different fixatives and of proteolytic digestion.
Authors: Dell'Orto P , Viale G , Colombi R , Braidotti P , Coggi G
Published: April 2011

The postembedding immunoperoxidase staining technique for the localization of immunoglobulins (light and heavy chains) and of lysozyme has been successfully applied to epoxy-embedded human lymph nodes, after removal of the resin. Glutaraldehyde-containing fixatives appear to be suitable for the immunohistochemical localization of human immunoglobulins and lysozyme, provided that the masked antigenicity of these proteins is recovered by proteolytic digestion of the tissue sections using 0.4% pepsin or 0.1% trypsin. Nonglutaraldehyde-containing fixatives allow the immunolocalization of human immunoglobulins without any enzymatic pretreatment. This study shows that tissues routinely fixed in glutaraldehyde and embedded for ultrastructural investigations are actually suitable for immunohistochemical studies on human immunoglobulins and lysozyme.
Characterization of epithelial elements in human major salivary glands by functional markers: localization of amylase, lactoferrin, lysozyme, secretory component, and secretory immunoglobulins by paired immunofluorescence staining.
Authors: Korsrud F R , Brandtzaeg P
Published: April 2011

Human parotid and submandibular glands were studied by paired immunofluorescence staining, including a variety of combinations of fluorochrome conjugates with contrasting colors. Lactoferrin (Lf), secretory component (SC), and particularly amylase were demonstrated in serous acinar cells of both glands. In addition, lysozyme (Ly) was present in some acini, although mainly located in intercalated ducts where Lf was also most commonly seen. SC was present in acini, intercalated ducts, and striated ducts but not in large collecting ducts. Staining for SC was generally faint but increased in intensity at the cell periphery and particularly at the luminal face of striated duct cells. Immunoglobulin (Ig) A--and IgM when detectable--showed an epithelial distribution similar to that of SC, in accordance with the known secretory properties of these two Ig classes. Conversely, IgG was not present in epithelial cells, despite its high extravascular concentrations. Mucous epithelial elements did not show unequivocal staining for any of the proteins studied. Formaldehyde fixation, combined with pronase treatment of tissue sections and prolonged exposure (20 hr) to antibody, enhanced markedly the staining intensity for lysozyme; ethanol fixation and 30-min incubation with conjugates generally afforded better localization of the other epithelial components.
Distribution of myoepithelial cells and basement membrane proteins in the resting, pregnant, lactating, and involuting rat mammary gland.
Authors: Warburton M J , Mitchell D , Ormerod E J , Rudland P
Published: April 2011

Using antisera to specific proteins, the localization of the rat mammary parenchymal cells (both epithelial and myoepithelial), the basement membrane, and connective tissue components has been studied during the four physiological stages of the adult rat mammary gland, viz. resting, pregnant, lactating, and involuting glands. Antisera to myosin and prekeratin were used to localize myoepithelial cells, antisera to rat milk fat globule membrane for epithelial cells, antisera to laminin and type IV collagen to delineate the basement membrane and antisera to type I collagen and fibronectin as markers for connective tissue. In the resting, virgin mammary gland, myoepithelial cells appear to form a continuous layer around the epithelial cells and are in turn surrounded by a continuous basement membrane. Antiserum to fibronectin does not delineate the basement membrane in the resting gland. The ductal system is surrounded by connective tissue. Only the basal or myoepithelial cells in the terminal end buds of neonatal animals demonstrate cytoplasmic staining for basement membrane proteins, indicating active synthesis of these proteins during this period. In the secretory alveoli of the lactating rat, the myoepithelial cells no longer appear to form a continuous layer beneath the epithelial cells and in many areas the epithelial cells appear to be in contact with the basement membrane. The basement membrane in the lactating gland is still continuous around the ducts and alveoli. In the lactating gland, fibronectin appears to be located in the basement membrane region in addition to being a component of the stroma. During involution, the alveoli collapse, and appear to be in a state of dissolution. The basement membrane is thicker and is occasionally incomplete, as also are the basket-like myoepithelial structures. Basement membrane components can also be demonstrated throughout the collapsed alveoli.
A stable propidium iodide staining procedure for flow cytometry.
Authors: Deitch A D , Law H , deVere White R
Published: April 2011

A propidium iodide (PI) staining procedure is described in which 50 micrograms/ml PI in 10(-2) M Tris, pH 7.0, with 5 mM MgCl2 is used to stain murine erythroleukemia cells (MELC) grown in suspension culture as well as single cell suspensions derived from rat kidney adenocarcinoma and human prostatic carcinoma. Specificity of staining of nuclear DNA is achieved by enzymatic removal of RNA using RNAse in the staining solution. Virtually identical histograms, with the same G1 peak height and closely similar coefficients of variation (CVs), are obtained using a wide range of RNAse concentrations on replicate samples of MELC if the incubation times are sufficiently prolonged when employing the lower enzyme concentrations. For 1 mg/ml RNAse on logarithmically growing MELC, 30 min incubation at 37 degrees C is needed to obtain a maximum G1 peak height and optimal CV and there is no significant change in the histogram if the incubation is prolonged to 4 hr. For every 4-fold decrease in RNAse concentration, the incubation time at 37 degrees C must be doubled to obtain the same maximal G1 peak height and optimal CV. Unfixed cell preparations, whether derived from suspension or monolayer cultures or from solid tumors, are stable for 2 or more weeks if stored at 4 degrees C between flow cytometric analyses and histograms are usually only minimally altered if the stained cell samples are stored for 1-2 months at 4 degrees C. Sample decay is associated with bacterial contamination. If sterile preparative techniques are used initially, subsequent contamination of the stained preparations may be minimized by adding sodium azide to the stained samples at 0.1% without influencing fluorescence intensity. Glycerine may be added to 10% and the samples slowly frozen for storage without altering DNA histogram shapes. The simplicity of sample preparation and the stability of the resulting stained cell samples makes this procedure suitable for repetitive comparative sampling of tissue and cell populations over prolonged time spans.
Distribution of sialoglycoconjugates on acinar cells of the mammalian pancreas.
Authors: Muresan V , Sarras M P , Jamieson J D
Published: April 2011

Using the sialic acid-specific lectin, limulin (LPA; from Limulus polyphemus hemolymph), the distribution and nature of sialoglycoconjugates on the surface of rat pancreatic cells has been investigated. Binding of rhodaminated LPA (Rh-LPA) or horseradish peroxidase-conjugated LPA (HRP-LPA) to fixed-frozen sections of adult rat pancreas resulted in intense linear staining of the apical surface of acinar cells with fainter staining on the basal but not the lateral cell surfaces. LPA binding was specific in that it could be abolished by 1) pretreatment of tissue sections with neuraminidase or periodic acid; 2) competition with sialic acid; and 3) incubation in Ca2+ -free buffers. Pretreatment of sections with proteases abolished LPA binding to the apical surfaces of acinar cells and also enhanced LPA binding to the lateral cell surface. Lipid extraction of sections following protease treatment markedly reduced LPA binding to the acinar cell periphery. These results suggest that LPA binding sites on the acinar cell apical surface may be primarily sialoglycoproteins, while those on the basolateral surfaces may consist in part of gangliosides. Electron microscopy of collagenase-dispersed acini exposed to HRP-LPA confirmed binding of LPA to the basal plasmalemma and, in addition, revealed staining of basal lamina when present. LPA binding to the acinar cell surface was not affected by digestion of tissue sections with hyaluronidase, heparinase, collagenase, or 6 M guanidine-HCl. Control experiments indicated that rat pancreatic secretory proteins contain undetectable amounts of sialoglycoproteins and thus that the apical localization of LPA is not due to adherent secretory proteins. Islets of Langerhans were always uniformly and heavily stained with LPA conjugates; this staining was protease insensitive. Appearance of LPA binding sites was examined on embryonic pancreatic epithelia. At day 15 of gestation, Rh-LPA stained the entire periphery of the epithelial cells, including the lateral cell surface, although more intense staining was already noted on the apical surface. This pattern persisted through day 17 of gestation, but by day 19 an adult staining pattern was observed with loss of staining of the lateral cell surfaces.
Cytochemical localization and measurement of aerobic 3,3'-diaminobenzidine oxidation reactions in photosynthetically grown Rhodospirillum rubrum.
Authors: Gierczak J S , Stevens F J , Pankratz H S , Uffen R L
Published: April 2011

Photoheterotrophically grown Rhodospirillum rubrum mutant C oxidized 3,3'-diaminobenzidine (DAB) by two aerobic reactions. One reaction was light dependent. The other respiratory reaction occurred in the dark and could be inhibited by 0.1 M KCN. DAB was not oxidized under anaerobic conditions. Cytochemical results, obtained by reacting viable cells with DAB, indicated that the two reactions were associated with different regions of the cellular membrane system. Under dark conditions, oxidized DAB was deposited along the cytoplasmic membrane. In the light, the cytochemical reagent accumulated at both the cytoplasmic and the intracytoplasmic photosynthetic membranes. DAB oxidation activity was measured by sensitive spectrophotometric and polarographic techniques. Under 880 nm actinic illumination, the rate of DAB oxidation was about 5.5-fold faster than in the dark. Determination of O2 consumption associated with the two DAB oxidation reactions showed that white light stimulated the rate of O2 uptake by about the same extent.
Differential uptake of peroxidase (HRP) and peroxidase-lectin (HRP-WGA) conjugate injected in the nodose ganglion of the cat.
Authors: Pugh W W , Kalia M
Published: April 2011

A comparison was made of the uptake and consequent axonal transport of peroxidase and peroxidase-lectin conjugate injected in low concentrations (0.167%) in the nodose ganglion of cats. At the light microscopic level horseradish peroxidase (HRP)-wheat germ agglutinin (WGA) intensely labeled only central terminal fields of vagal afferents (anterograde), while free HRP only labeled perikarya in vagal motor nuclei (passing retrograde). Low concentrations of these proteins, in addition to normal diffusion equilibria, permit a differential distribution of those species demonstrating some affinity for cell membranes. We attribute these differences in the uptake of HRP and HRP-WGA to the selective affinity of WGA for cell surface receptors (n-acetyl glucosamine) on the plasma membrane. This results in a greater number of WGA molecules coupled to HRP being internalized in any given endocytotic event compared to free HRP. The fractionation of efferent and passing fiber populations within a nodosal injection site can be discriminated with these different preparations.
Immunoelectron microscopic localization of immunoglobulins and complement in human renal glomeruli.
Authors: Rantala I , Helin H
Published: April 2011

A study was undertaken to evaluate some processing variables affecting the immunoelectron microscopic demonstration of immunoglobulins and complement (C3) in human glomeruli. Percutaneous biopsies were performed on 28 patients with various types of glomerulonephritis. Light microscopic, electron microscopic, and immunofluorescence examinations were performed by routine methods. For immunoelectron microscopy, fixation in paraformaldehyde (PA) or periodate-lysine-paraformaldehyde (PLP) was used. With the diffusion technique, using tissue chopper or cryostat sections, human immunoglobulin (Ig)G, IgA, IgM, and C3 were localized in glomeruli with peroxidase-labeled antisera. Using PLP and the tissue chopper sections, good ultrastructure was achieved. The antigens could be demonstrated in intramembranous, subepithelial, subendothelial, or mesangial immune deposits. Penetration of antibodies and quality of peroxidase reaction in the cryostat sections did not differ from that of the tissue chopper sections. Freezing and thawing, however, resulted in inferior morphology. If PA was used, the antigens could not be reliably demonstrated. The results of light microscopy, electron microscopy, and immunofluorescence microscopy were in good agreement with those from the immunoperoxidase procedure. The present study shows that PLP preserves well the antigenicity of human immunoglobulins and C3, resulting in good ultrastructure. PA fixation, on the contrary, caused a loss of antigenicity before an adequate ultrastructure could be achieved.
Brain clathrin: immunofluorescent localization in rat retina.
Authors: Bloom W S , Puszkin S
Published: April 2011

By indirect immunofluorescence, the major protein of coated vesicles and coated pits was localized in rat retina. Specific fluorescence was circumscribed to regions containing synaptic endings in high density such as the cell internal plexiform layer (IPL) and external plexiform layer (EPL). Fine dots or patches of fluorescence were seen in the EPL. Weaker fluorescence was observed in the narrow cytoplasmic strips surrounding the large nucleus of the ganglion cell, internal nuclear and external nuclear cell layers. There was no fluorescent staining in the retinal pigment epithelium, a layer where active endocytosis of the outer segment of the rods and cones take place. The high concentration of clathrin found in the IPL and EPL is indicative of active recycling of synaptic membrane concomitant to neurotransmitter release and receptor molecule turnover.
Cytochemical methods for the localization of adenylate cyclase. A review and evaluation of the efficacy of the procedures.
Authors: Cutler L S
Published: April 2011

The cytochemical procedures for localizing adenylate cyclase have been a source of controversy since their introduction. The importance of cyclic adenosine monophosphate (AMP), the product of adenylate cyclase's action on adenosine triphosphate (ATP), in cell biology is clear. Thus, the ability to localize this enzyme system reliably is an important tool in the study of various cellular functions. This report reviews the literature and presents a biochemical evaluation of the methods for localizing adenylate cyclase. The review and data presented serve to clarify many of the controversies surrounding this important cytochemical procedure. It is evident that although there are problems associated with localizing the enzyme, several valid procedures are currently available for the cytochemical localization of adenylate cyclase. In using these procedures, the effects of fixation and the capture agent on adenylate cyclase activity in the particular tissue being studied should be considered. Only repurified adenylyl imidodiphosphate [App(NH)p] should be used in the incubation medium. If care is taken, the use of these techniques can be of great value in the continued study of the role of cyclic nucleotides in cell biology.
Ultrastructural immunocytochemical localization of the phosphomannosyl receptor in Chinese hamster ovary (CHO) cells.
Authors: Willingham M C , Pastan I H , Sahagian G G
Published: April 2011

Using ultrastructural immunocytochemistry and antibodies directed against bovine liver phosphomannosyl (PM) receptor, we have localized the receptor in Chinese hamster ovary (CHO) cells. The majority of the receptor was found within the cell. Only a small fraction of the receptor was found on the surface and most of it was clustered in coated pits. Because these cells contain endogenous ligands for the receptor, it was not possible to determine if this clustered state was dependent on occupancy of the receptor. The bulk of the cell's receptor was found in the endoplasmic reticulum, nuclear envelope, and in the Golgi system. Most of the Golgi localization was associated with peripheral Golgi elements, suggesting a possible concentration of receptor in GERL. Very little receptor was found associated with mature lysosomes. PM receptor was also localized in structures that were identified as receptosomes by the presence of alpha 2-macroglobulin (alpha 2M)-gold, a ligand previously shown to enter CHO cells by the coated pit-receptosome pathway. This finding is consistent with the notion that during receptor-mediated endocytosis, receptors accompany ligand from the coated pit into the receptosome. The observation that the majority of the receptor was found in the endoplasmic reticulum and structures similar to GERL raises the possibility that the PM receptor plays an important role in compartmentalization of lysosomal enzymes in the GERL system.
Ultrastructural localization of 5'-nucleotidase in guinea pig neutrophils based upon the use of cerium as capturing agent.
Authors: Robinson J M , Karnovsky M J
Published: April 2011

A cytochemical method for 5'-nucleotidase localization in which cerium serves as the capture agent in order to enzymatically detect liberated inorganic phosphate has been developed. The method has been established in cell-free model systems and in guinea pig neutrophils where 5'-nucleotidase is restricted to the plasmalemma as an ectoenzyme. This cerium-based method gives better results for ultrastructural localization of 5'-nucleotidase than conventional lead-based methods.
Ultrastructural localization of several phosphatases with cerium.
Authors: Robinson J M , Karnovsky M J
Published: April 2011

Cerium ions have been used as the capture agent for inorganic phosphate released during the enzymatic hydrolysis of phosphate-containing substrates by a variety of phosphatases. Cerium phosphate reaction product accumulation is proportional to the amount of enzyme present in a cell-free model system. Ultrastructurally, cerium phosphate reaction product appears as a very fine electron-dense precipitate. Cerium appears to be a better capture agent for inorganic phosphate than lead in that reaction product is usually more uniform and more consistently reproducible when cerium is used. Furthermore, nonspecific deposits of reaction product that are commonly encountered in lead-based phosphatase reactions are virtually nonexistent when cerium is the capture agent.