bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of techniques improving the resolution of standard Coulter cell sizing systems.
Authors: Von Behrens W , Edmondson S
Published: April 2011

Latex spheres with nominal diameters of 1.857, 2.02, 3.5, 5.2 and 9.69 mum were used to compare improvement in Coulter resolution gained (a) by a simple hydrodynamic focusing modification and (b) by Coulter edit and high resolution circuits. Since pulse size profiles of focused 9.69-mum spheres appeared quasimonodisperse [geometric SD (GSD) congruent to 0.016 congruent to (1 + Arithmetic SD/arithmetic mean)], unfocused profiles of these particles approximated the error function resulting from inhomogeneities in the electric and hydrodynamic sensing-zone fields. As expected, this field error function differed systematically with differences in orifice diameters (30, 50, 70 and 100 mum) and length to diameter ratios (0.75 or 1.2), the error being reduced slightly by the edit circuit and considerably by the high resolution circuit. Data on the 1.857- and 2.02-mum spheres (focused GSDs congruent to 1.06) and 3.5- and 5.2-mum spheres (focused GSDs congruent to 1.6) indicated that the field error affected closely similar particles similarly but its influence on the unfocused population means and variances differed in the GSD regions of 1.06 and 1.60.
Cell discrimination by multiangle light scattering.
Authors: Loken M R , Sweet R G , Herzenberg L A
Published: April 2011

Measurement of the light scattered by biological cells as a function of scattering angle provides information that can be correlated with cell type. Two flow systems that provide multiangle scattering data from cells have been constructed and tested. The first utilizes two narrow-aperture detectors positioned at different angles; the second utilizes the motion of the cell to generate complete scatter patterns of individual cells over a 67 degrees range of scattering angle.
Pulse-height light-scatter distributions using flow-systems instrumentation.
Authors: Mullaney P F , Crowell J M , Salzman G C , Martin J C , Hiebert R D , Goad C A
Published: April 2011

Several laboratories have recently been making light-scatter measurements on cells and other particles using flow-systems instrumentation. We at the Los Alamos Scientific Laboratory, as well as others, have obtained multimodal pulse-height distributions in certain angular regimes from particles of supposedly uniform characteristics. Because it was assumed that multimodal distributions implied characteristics of multivalue, the accuracy of such data has been doubted. In the present work, pulse-height distributions anticipated on the basis of exact electromagnetic theory were calculated for particles of known characteristics. These calculated pulse-height distributions agree quite well with those obtained experimentally. Physical optics form the basis for the explanation of the complex pulse-height distributions obtained experimentally. However, the results of this study show that certain cautions are necessary in the interpretation of light-scatter data presented in this manner.
Slit-scan cytofluorometry. Basis for automated prescreening of urinary tract cytology.
Authors: Cambier M A , Christy W J , Wheeless L L , Frank I N
Published: April 2011

A study was undertaken to assess the applicability of the slit-scan technique to automated prescreening of urinary tract cytology. Cells from voided and catheterized urines were stained with acridine orange and measured on a static cell slit-scan cytofluorometer. Analysis of data from the specimens indicates that nuclear fluorescence alone appears adequate for recognition of abnormal specimens. Remaining problems in the automation of urinary tract cytology prescreening are discussed.
Purification of the chromosomes of the Indian muntjac by flow sorting.
Authors: Carrano A V , Gray J W , Moore D H , Minkler J L , Mayall B H , van Dilla M A , Mendelsohn M L
Published: April 2011

Metaphase chromosomes were isolated from a male Indian muntjac cell line, were stained with ethidium bromide and were analyzed by flow microfluorometry to establish a deoxyribonucleic acid (DNA)-based karyotype. Five major peaks were evident on the chromosomal DNA distribution corresponding to the five chromosome types in this species. The amount of DNA in each chromosome was confirmed by cytophotometric measurements of intact metaphase spreads. The five chromosome types were separated by flow sorting at rates up to several hundred chromosomes per second. The sorted chromosomes were identified by morphology and by Giemsa banding patterns. The automsomes, Numbers 1, 2 and 3, and the X + 3 composite chromosome were separated with a high degree of purity (90%). The centromere region of the X + 3 chromosome was fragile to mechanical shearing, and during isolation a small proportion of these chromosomes broke into four segiments: the long arm, the short arm, the short arm plus centromere and the centromere region. A large fraction of the constitutive heterochromatin of this species is present in the centromere region of the X + 3 chromosome and in the Y chromosome; these two regions possess similar amounts of DNA and therefore sort together. Chromosome flow sorting is rapid, reproducible and precise; it allows the collection of microgram quantities of purified chromosomes.
Automated analysis of antigen-stimulated lymphocytes.
Authors: Cassidy M , Yee C , Costa J
Published: April 2011

Human peripheral blood lymphocytes were stimulated with phytohemagglutinin and tuberculin-purified protein derivative to determine if flow microfluorometric techniques had the sensitivity to detect high and low levels of blastogenic response. The deoxyribonucleic acid content of the lymphocytes was analyzed after culture. Blastogenic response was found to be measurable both sensitively and reproducibly. Tritiated thymidine incorporation measurements were also made. Preliminary experiments using inactivated Herpesvirion (type I) as a stimulant showed blastogenic response detectable by flow microfluorometric deoxyribonucleic acid measurements. Refinements of this technique may prove to be useful in the study of lymphocyte response to viral antigens in patient populations.
Quantitation of fluorescence fading phenomena for identifying intracellular biopolymers.
Authors: West S S , Golden J F , Menter J M , Love L D
Published: April 2011

The fading behavior of the 670 nm fluorescence emission band produced by unfixed rat mast cells stained with acridine orange (AO) has been found to be in excellent agreement with the behavior predicted by second order chemical kinetics. The reciprocal of fluorescence intensity plotted against time yields a straight line. When due account is taken of dye/cell ratio and the intensity of fluorescence-exciting radiation, Io (measured with the standard phosphor particle), the slope of this straight line is a constant, k'', which is independent of dye/cell ratio and Io. k'' differs from the second order photochemical rate constant by a constant factor. The fading of a given AO-biopolymer complex is described by a particular value of k''. Two values of k'' have been found for rat mast cell granules, indicating the presence of two different AO-biopolymer complexes. Fading of fluorescence may serve to identify particular intracellular biopolymers in individual cells even when present in a heterogeneous population.
Quantitation of the rate-zonal sedimentation spectrum.
Authors: Tolles W E
Published: April 2011

Rate-zonal sedimentation of cells from the female genital tract has been employed as a means of separating many of the different cellular components from each other. Ultra-slow streak photography of forward scattered light from the sedimentation spectrum has been used as a monitor of the success of the individual sedimentation maneuver. The presence of convention currents vitiates the separation potential of the technique, and this is readily detected by the photographic monitor. In addition, these photographs clearly indicate the positions of bands of cellular entities of different sedimentation velocities. Further study of the quantitative aspects of the light scattered from each part of the spectrum reveals that it possesses a quantitative representation of the concentration of cells at each level of the spectrum. The photographs and densitometric scans support these expectations. To avoid the limitations of film as a data-logging medium and to obtain real-time system utilization, a direct photoelectric scanner has been assembled. Preliminary tests indicate ready feasibility.
Automatic leukocyte classification using cytochemically stained smears.
Authors: Tycko D H , Anbalagan S , Liu H C , Ornstein L
Published: April 2011

A leukocyte classification algorithm suitable for automated differential counting has been developed for blood smears stained with a new three-component cytochemical stain which has relatively narrow absorption bands centered at 460, 540 and 640 nm, respectively. The classification procedure is the result of a pattern recognition experiment using a sample of 223 leukocytes distributed evenly over the five normal cell types. The basic data for each cell were three digital microscopic images obtained with narrow band illumination at the above central wavelengths using a TV-digitizer system interfaced to a PDP-15 computer. The classification algorithm involves a sequential decision procedure utilizing five pattern features computed from the intensity histograms of the green and blue digital images. Thus the number of arithmetic operations and the number of computer memory words necessary to perform the classification into one of the five normal white blood cell types are both proportional to n where n is the number of gray levels into which the intensity scale is divided. In this experiment, n equals 256. Comparison of our results with work of others on smears prepared with Romanowski-type stains indicates that such narrow-band, spectrally well separated cytochemical multiple stains can permit the use of algorithms which are approximately ten times faster.
Deoxyribonucleic acid replication in single cells and chromosomes by immunologic techniques.
Authors: Gratzner H G , Pollack A , Ingram D J , Leif R C
Published: April 2011

Antibodies to 5-bromodeoxyuridine (BrdU) or iododeoxyuridine may be used to identify cells or regions of chromosomes in which de novo deoxyribonucleic acid synthesis has occurred. The antibodies to BrdU were produced in rabbits by injection of the antigen, a conjugate between bovine serum albumin and bromouridine (BrU), or iodouridine. Specific antibodies were produced by affinity chromatography on AH-Sepharose 4B to which had been coupled BrU. Anti-BrU cross-reacts with iodeodeoxyuridine. Indirect antibody techniques have been used to monitor deoxyribonucleic acid synthesis in nuclei; anti-BrdU treatment was followed by goat anti-rabbit immunoglobulin G labeled with either fluorescein or horseradish peroxidase. By use of these techniques, labeling indices were determined in cell cultures which had been pulsed with 3H-BrdU. The immunologic technique compared favorably with the autoradiographic methods performed concurrently on the same cultures. Metaphase chromosomes from synchronous CHO cell which had been pulse labled with BrdU at different time intervals during S phase were subjected to these immunologic procedures. Chromosome banding was observed with both the fluoresence and peroxidase methods. Chromosomes from cells not containing BrdU did not exhibit banding.
Spectral studies on 33258 Hoechst and related bisbenzimidazole dyes useful for fluorescent detection of deoxyribonucleic acid synthesis.
Authors: Latt S A , Stetten G
Published: April 2011

Absorption, fluroescence and circular dichroism measrements on 33258 Hoechst-deoxyribonucleic acid (DNA) complexes are consistent with the existence of two types of dye-binding interactions. One type, which persists at elevated solution ionic strength, is highly specific for adenine-thymine-rich DNA. Dye bound under this condition exhibits efficient fluorescence and strong optical activity. A less specific, largely electrostatic interaction is associated with less intense fluorescence and weaker optical activity. The fluorescence of 33258 Hoechst and several other bisbenzimidazole dyes is less when bound to poly(deoxyadenylate-5-bromodeoxyuridylate) than when bound to poly(deoxyadenlyate-deoxythymidylate). Quenching of 33258 Hoechst fluorescence can also be used to detect biosynthetic incorporation of 5-bromodeoxyuridine into the DNA of living cells. This property of 33258 Hoechst should allow fluorescence-activated cell and chromosome sorting according to the extent of DNA synthesis, providing a bridge between biochemical and cytologic analyses of processes related to DNA replication.
Mouse ovarian alkaline phosphatase activities that respond to gonadotropins: histochemical and biochemical studies.
Authors: Bramley T A , Kent J
Published: April 2011

Alkaline phosphatase activity was measured in whole ovarian homogenates from pre-pubertal mice of different ages, with and without prior injection of human chorionic gonadotropin. Alkaline phosphatase activity was also scored in the different cell types in sections of similar ovaries, using two distinct histochemical procedures. The results from those methods differed. Biochemical studies indicated the presence of three distinct alakaline phosphatase activities: I and Ib, both optimal at pH 10.4 and with similar substrate requirements and inhibitor sensitivities (phosphatase I being characteristic of unstimulated ovaries and Ib of ovaries stimulated with human luteinizing hormone or human chorionic gonadotropin), and phosphatase II, optimal at pH 9.4, with different substrate requirements and inhibitor sensitivities. The differences observed using the histochemical procedures can probably be accounted for by the effects of different incubation conditions on the activities of these three enzymes.
Cytochemical study of the distribution of adenosine triphosphatase in the pancreas of the dog.
Authors: Koenig C S , Santelices L C , Vial J D
Published: April 2011

Dog pancreatic tissue, incubated in a modified Wachstein-Meisel medium, showed two different adenosine triphosphatase activities. One of them is located at the apical border of the cells lining the intralobular ducts and of the centroacinar cells and is stimulated by HCO3-, depressed by SCN- and OCN- and completely abolished by CN-. The other is located at the intracellular clefts of the epithelium lining the interlobular ducts and is stimulated by Mg++. These findings correlate well with the results of incubation of homogenates of fresh and fixed tissues. Their significance with respect to the role of different segments of the duct system in the formation of the pancreatic juice is discussed.
An immunocytochemical study of TSH beta storage in rat thyroidectomy cells with and without D or L thyroxine treatment.
Authors: Moriarty G C , Tobin R B
Published: April 2011

Binding sites to the beta chain of thyroid stimulating hormone (TSH) were localized in pituitaries of thyroidectomized rats. Immunocytochemical staining was observed in hypertrophied TSH cells ("thyroidectomy cells") and primarily located in dilated rough endoplasmic reticulum. Staining was also found on the few secretory granules and on some of the intracisternal granules. Some of the thyroidectomy cells stained intensely, while others exhibited very little staining. When thyroidectomized rats were treated with thyroxine 4 days before death, the TSH cells contained more secretory granules, and the intracisternal granules were larger and more numerous. L-thyroxine was 10 times as potent as D-thyroxine in promoting the build-up of granules. Both types of granules stained intensely.
Use of actinomycin D for the specific quenching of fluorescence of deoxyribonucleic acid in cells stained with acridine aminoderivatives.
Authors: Zelenin A V , Kirianova E A , Kolesnikov V A , Stepanova N G
Published: April 2011

Actinomycin D specifically quenches the fluorescence of acridine orange and quinacrine bound to deoxyribonucleic acid in cytologic preparations, but does not change the fluorescence of these fluorochromes bound to RNA. The following fluorescence-cytochemical applications of techniques based on these findings can be suggested: (a) distinction between deoxyribonucleic acid and ribonucleic acid; (b) detection of double-stranded virus ribonucleic acid; (c) approximate estimation of the lengths of A-T sequences in deoxyribonucleic acid molecules.
Immunohistochemical localization of melatonin in the rat Harderian gland.
Authors: Bubenik G A , Brown G M , Grota L J
Published: April 2011

Using fluorescence and double antibody techniques, melatonin was localized immunohistologically in the secretory cells of the Harderian gland of mature male rats. The presence and quantity of melatonin in the acinar cells seem to correlate with the amount of porphyrins inside the lumen. The specificity was proven by disappearance of yellow fluorescence after saturation of antibody with melatonin or after use of nonspecific antibody only.
Ultrastructural immunocytochemical characterization of the thyrotroph in rat and human pituitaries.
Authors: Moriarty G C , Tobin R B
Published: April 2011

The beta chain of thyroid stimulating hormone (TSH) was localized in normal rat and human pituitaries with the unlabeled antibody--peroxidase-antiperoxidase complex technique and antisera to rat or bovine TSH beta. Araldite embedded tissues, immunoreactivity of TSH was best preserved after fixation with 1% glutaraldehyde or picric acid formaldehyde. In the TSH cells, the immunocytochemical stain was located on granules. However, there was a variation in staining of individual granules, and among the population of granules. Rat TSH cells were ovoid or angular to stellate, and contained granules ranging in size from 60-175 nm. The human TSH cell was polyhedral, and contained scattered granules of 150-300 nm diameter. In both species, granule distribution was either in 1-2 rows at the periphery, or scattered throughout the cytoplasm with some concentration in cytoplasmic processes. TSH cells in female rats in estrous contained more granules than those of other stages. TSH cells were distinguished from adrenocorticotropic hormone cells and luteinizing hormone cells on the basis of granule size and distribution, and cell shape.
A new histochemical method for the identification and visualization of both side chain acylated and nonacylated sialic acids.
Authors: Culling C F , Reid P E , Dunn W L
Published: April 2011

A new histochemical method is described for the differentiation of mucins that utilizes two different Schiff reagents and allows single section identification of side chain O-acylated, and nonacylated, sialic acids in contrasting colors. In the event of mucins containing only one type of sialic acid, it may allow their specific identification (e.g., C7 or C8 side chain O-acylated). It has been shown to be useful in the identification of some metastases from adenocarcinomas of colon (where the primary is potassium hydroxide/periodic acid-Schiff positive) and should prove of great value in the investigation of diseases of the gastrointestinal tract and particularly those of the colon. It should also be valuable in the general field of epithelial mucin histochemistry, particularly for those mucins of the salivary and parotid glands, etc.
Lasar flow cytophotometric immunoperoxidase detection of herpes simplex virus type 2 antigens in infected cultured human cells.
Authors: Leary J F , Notter M F , Todd P
Published: April 2011

Human cells in culture (HEp-2) were infected with herpes simplex virus type 2 (HSV-2) at multiplicities of infection varying from 0.2 to 10, and fixed 6, 12, 18 and 24 hr after infection. Infection-related antigens were detected by an indirect double antibody (peroxidase conjugated goat anti-rabbit to rabbit anti-herpes simplex virus type 2) immunoenzymatic staining reaction that rendered infection-related antigens visible by light microscopy. A corresponding series of laser flow cytophotometric experiments yielded reproducible large-angle (1-19 degrees) laser-light scattering distributions that depended upon multiplicities of infection and the location of the infection-related antigens in the infected cells.
The identification of eosinophil colonies in soft-agar cultures by differential staining for peroxidase.
Authors: Zucker-Franklin D , Grusky G
Published: April 2011

There has been a need to easily quantitate the incidence of eosinophil colonies within soft agar cultures. This has been realized by layering of the agar with benzidine dihydrochloride that permits detection of peroxidase activity in cells. Eosinophil colonies can be specifically identified by the addition to the substrate of potassium cyanide, an inhibitor of enzyme activity in neutrophils and monocytes. The enumeration of eosinophil colonies can be accomplished by scanning fresh or embedded cultures with low power magnification.
Ultrastructural cytochemistry of p-N,N-dimethylamino-beta-phenethylamine (DAPA) oxidation reactions. "activation" and inhibition studies.
Authors: Shannon W A , Wasserkrug H L , Seligman A M
Published: April 2011

The oxidation of p-N,N-dimethylamino-beta-phenethylamine (DAPA) by amine oxidase(s) (AO), i.e., diamine oxidase (DAO), monoamine oxidase (MAO) and/or possibly other oxidases, has been previously demonstrated. This study reports the results of variations in fixation procedures and the incorporation of a series of possible "activators" and inhibitors into the DAPA oxidase (DAPAO) and DAPAO-BSPT [2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl) tetrazolium chloride] reaction media in an attempt to elucidate the oxidase(s) involved. Results of these studies are indicative of at least two different oxidases acting preferentially on one or the other of the two systems. The presence of MAO, especially in unfixed tissue, and DAO, especially in fixed tissue, is denoted and that of other oxidase(s) is connoted.
Plasma membrane localization of alkaline phosphatase in HeLa cells.
Authors: Lin C W , Sasaki M , Orcutt M L , Miyayama H , Singer R M
Published: April 2011

The localization of alkaline phosphatase in HeLa cells was examined by electron microscopic histochemistry and subcellular fractionation techniques. Two monophenotypic sublines of HeLa cells which respectively produced Regan and non-Regan isoenzymes of alkaline phosphatase were used for this study. The electron microscopic histochemical results showed that in both sublines the major location of alkaline phosphatase is in the plasma membrane. The enzyme reaction was occasionally observed in some of the dense body lysosomes. This result was supported by data obtained from a subcellular fractionation study which showed that the microsomal fraction rich in plasma membrane fragments had the highest activity of alkaline phosphatase. The distribution of this enzyme among the subcellular fractions closely paralleled that of the 5'-nucleotidase, a plasma membrane marker enzyme. Characterization of the alkaline phosphatase present in each subcellular fraction showed identical enzyme properties, which suggests that a single isoenzyme exists among fractions obtained from each cell line. The results, therefore, confirm the reports suggesting that plasma membrane is the major site of alkaline phosphatase localization in HeLa cells. The absence of any enzyme reaction in the perimitochondrial space in these cultured tumor cells also indicates that the mitochondrial localization of the Regan isoenzyme reported in ovarian cancer may not be a common phenomenon in Regan-producing cancer cells.
Histochemical localization of potassium-stimulated P-nitrophenylphosphatase activity in the somatosensory cortex of the rat.
Authors: Stahl W L , Broderson S H
Published: April 2011

Potassium-stimulated p-nitrophenylphosphatase (K+-pNPPase) activity was investigated in rat somatosensory cortex where 64-88% of enzymatic activity survived 5-10 min of fixation with 3% formaldehyde in 0.1 M cacodylate buffer, pH 7.4. Potassium-stimulated activity was inhibited by 1-10 mM ouabain. Levamisole (1.7 mM) inhibited brain alkaline phosphatase activity, facilitating the detection of K+-pNPPase activity. Strontium (10-20 mM) inhibited enzymatic activity by 38-75%. In parallel histochemical studies reaction product was found in strata, with cortical layers 2, 3, 4 and the outer portion of 5 containing the heaviest deposits. Highly reactive, vertically oriented, large diameter fibers were seen as groups between the outer portion of layer 5 and the pail surface. These fibers apparently arborize in the superficial layers. Smaller fibers were also positive and were oriented in various planes. The highest density of smaller, positive fibers occurred in layers 2 through 5. All positive fibers appeared to be axons or dendrites. Reaction product was not heavily concentrated in neuron perikarya or in glial elements. Sections did not contain reaction product when incubated in media lacking K+ or containing ouabain. The convergence of data from parallel histochemical and biochemical approaches supports the conclusion that the reactivity localized in the cerebral cortex represented the site of K+-pNPPase, a known component of the Na+,K+-adenosine triphosphatase complex. Neuronal processes demonstrated the highest enzymatic activity and may be most important in the active transport of Na+ and K+ in somatosensory cortex.
Immunohistochemical localization of hypothalamic hormones.
Authors: Pelletier G , Leclerc R , Dubé D
Published: April 2011

Using an immunoperoxidase technique at the light and electron microscope levels, the localization of somatostatin and luteinizing hormone-releasing hormone (LH-RH) has been performed in the brain, as well as the pancreas and stomach. The following generalizations may be drawn on the localization of LH-RH and somatostatin. (a) LH-RH and somatostatin are contained in axons and nerve endings of the median eminence and organum vasculosum of the lamina terminalis. (b) LH-RH and somatostatin are present in different nerve endings. (c) In the subcommissural organ, subfornical organ and area postrema, the two neurohormones are present in the cytoplasm of ependyman and subependymal cells. (d) In the endocrine pancreas and gastric mucosa, somatostatin is localized within the secretory granules of specific endocrine cells.
Immunocytochemical detection of casein and casein-like proteins in human tissues.
Authors: Pich A , Bussolati G , Carbonara A
Published: April 2011

By means of specific immunochemical methods, material reacting with anti-human casein antisera has been detected in various human tissues and mainly in ductules of the breast, in the sebaceous and sweat glands of the skin, in the bronchial epithelium and glands, at the surface of some alveoli of the lung, in the exocrine pancreas, in the glands of the endometrium in proliferative phase and in the distal and collecting tubules of the kidney. The exact chemical nature and the physiological significance of the proteins present in extramammary sites and reacting with anti-casein antibodies are not clear; it appears, however, that casein might be a protein not specific of the breast, and that casein-like material might be present in other areas and mainly devoted to exocrine secretion. The interest of these findings is also related to the presence of proteins reacting with anti-human casein antisera in carcinomas of the lung, the endometrium and the gastrointestinal tract.
Precautions in the application of immunohistochemical techniques to tissues of the pig hypothalamo-neurohypophysial system.
Authors: Watkins W B
Published: April 2011

Immunohistochemical procedures were used to localize neurophysin in the hypothalamo-neurohypophysial axis of the domestic pig. The topographical distribution of neurophysin as revealed by the immunofluorescence "sandwich" technique was similar to that found when either the immunoglobulin-peroxidase bridge method or the peroxidase-labeled gamma-globulin technique was employed. However, application of the peroxidase-anti-peroxidase (PAP) complex procedure resulted in nonspecific staining of the magnocellular structures. This phenomenon was attributed to the action of PAP on the tissue and after screening a number of other vertebrate species was found to be unique to the pig. Minimal nonspecific binding of the PAP could be achieved either by reducing the reaction time of PAP to 5 min or, by the addition of 1% (v/v) normal serum to all reagents and wash solution. That the PAP-binding protein is a component of the hypothalamo-neurohypophysial axons is discussed.
Immuno-electron-cytochemical localization of the somatostatin cells in the human antral mucosa.
Authors: Canese M G , Bussolati G
Published: April 2011

Immuno-cytochemical methods were used to identify, in light and electron microscopy, the somatostatin-containing cells of the human antral mucosa. By means of immunoperoxidase and immunofluorescence methods sequentially applied on the same section, it was shown that the somatostatin cells are distinct from the gastrin cell population; these two endocrine cell types are often closely related. On ultrathin sections from aldehyde-fixed. Epon-araldite embedded tissues, the site of storage of somatostatin was localized with the peroxidaseantiperoxidase complexes technique, after removal of the resin by means of sodium ethoxide. This procedure represents a new technical approach to the use of electron-cytochemical techniques. The results indicate that somatostatin, a growth hormone release inhibiting factor, is localized in the endocrine granules of the D cells.
Peroxidase and gold complexes of lectins for double labeling of surface-binding sites by electron microscopy.
Authors: Roth J , Wagner M
Published: April 2011

Double labeling experiments were performed for visualization of the binding sites of Concanavalin A and anti-AHel (the lectin from Helix pomatia). The anti-AHel was labeled with the colloidal gold whereas the membrane bound Concanavalin A was demonstrated by an affinity technique using horseradish peroxidase. The two markers used could be clearly distinguished electron microscopically. The specificity of the cell surface double labeling was demonstrated in the control experiments. A topological distinct localization of the both lectin-binding sites is evident.
Protein A-peroxidase: a valluable tool for the localization of antigens.
Authors: Dubois-Dalcq M , McFarland H , McFarlin D
Published: April 2011

Protein A of Staphylococcus aureus has been conjugated to horseradish peroxidase and used in an indirect immunolabeling technique to visualize membrane and viral antigens. The same Protein A-peroxidase conjugate was used with antisera from five different species. Using this indirect test, membrane markers for T and B lymphocytes were labeled with a greater specificity than when peroxidase conjugated anti-immunoglobulin was used in the second step. Viral antigens on cells infected with measles, vesicular stomatitis, herpes or visna virus, respectively, were also stained in the protein A-peroxidase indirect test with a greater specificity than indirect method using anti-immunoglobulin. Paired preparations were examined in the light and electron microscope. Ultrastructural analysis showed that the protein A-peroxidase conjugate penetrated well through fixed viral membranes and resulted in fine resolution of antigenic sites.
Adsorption of horseradish peroxidase, ovomucoid and anti-immunoglobulin to colloidal gold for the indirect detection of concanavalin A, wheat germ agglutinin and goat anti-human immunoglobulin G on cell surfaces at the electron microscopic level: a new method, theory and application.
Authors: Geoghegan W D , Ackerman G A
Published: April 2011

A method is described for the adsorption of selected macromolecules to colloidal gold which is then used as an electron dense marker for the indirect detection of specific cell surface molecules. Membrane bound concanavalin A, which binds specific sugars on horseradish peroxidase, and wheat germ agglutinin, which binds specific sugars on ovomucoid are detected indirectly with gold labeled horseradish peroxidase and ovomucoid, respectively. Goat anti-human IgM on blood lymphocytes is detected with gold labeled rabbit anti-goat IgG. In the preparation of colloidal gold labeled proteins, the problems of flocculation of colloidal gold by proteins and nonadsorption of proteins to colloidal gold, are solved through a combination of concentration of protein and pH variable adsorption isotherms, which allows one to determine the conditions for adsorption of proteins to colloidal gold. Adsorption is pH dependent, the pH conditions correlating with the isoelectric point(s) of the major protein fraction(s); adsorption is influenced by interfacial tension, solubility and by the electrical charge on the molecules. Colloidal gold is inexpensive and preparation of a useful label is rapid, reproducible and the results easily quantitated from electron micrographs.
Immunoelectron microscopic visualization of neurohypophyseal hormones: evaluation of some tissue preparations and staining procedures.
Authors: Van Leeuwen F W
Published: April 2011

To obtain optimal electron microscopical localization of vasopressin in the rat neurohypophyses two immunocytochemical staining procedures and several tissue treatments were evaluated. The peroxidase-antiperoxidase technique allowed greater dilution of the first antibody than the indirect method using a commercial peroxidase conjugate. This appeared crucial for the dilution-dependent specificity in the localization of vasopressin. Immersion fixation with formalin gave better results than those obtained with perfusion fixation with glutaraldehyde-paraformaldehyde (resulting in similar preservation of immunoreactivity) and freeze substitution (showing the best preservation of immunoreactivity). However, these latter two tissue fixation methods resulted in less than optimal preservation of general ultrastructure than immersion fixation in formalin alone. Immersion fixation with glutaraldehyde-paraformaldehyde followed by OsO4 improved ultrastructural detail, but immunoreactivity decreased considerably. Fixation with paraformaldehyde-picric acid resulted in poorest preservation of morphologic detail. Immunoreactivity was similar in both Epon 812 and Araldite 6005 embedded tissue.
Purification of commercial acriflavine by Sephadex LH-20 column chromatography.
Authors: Levinson J W , Maher V M , McCormick J J
Published: April 2011

Acriflavine supplies commercially was purified by removing contaminating proflavine by chromatography on a column of Sephadex LH-20 eluded with 65% methanol. The purified acriflavine, recovered in a yield that was 52% of the theoretical maximum, had a visible absorption maximum of 452 nm with a band width of 47 nm. These spectral properties agreed with those of acriflavine purified by precipitation, repeated recrystallization and chromatography on aluminum oxide, which results in a yield of 13% at best.
A technique for the immunohistochemical localization of prostaglandin E.
Authors: Van Orden D E , Klemm J M , Redick J A
Published: April 2011

Prostaglandin E (PGE) has been localized via the unlabeled antibody technique in freeze-dried and ethanol-fixed cryostat sections. Discrete perivascular and stromal localization was present in the uterus prepared by the method presented, but not in classically fixed specimens. Absorption of the anti-PGE by addition of free PGE was ineffective; whereas, removal of PGE-reactive antibodies from the anti-serum was effectively accomplished with an Affigel-101-PGE immunoadsorbant column.
Two-wavelength cytophotometry: the choice of the wavelengths from a practical point of view.
Authors: Van Oostveldt P , Boeken G
Published: April 2011

Methods in use for the choice of the two-wavelengths and the effect of a faulty choice of the two wavelengths in the two-wavelength cytophotometric method of Patau and Ornstein are studied by means of a computer simulation and in practice upon pararosaniline-Schiff stained nuclei. A simplification of the measuring strategy is proposed without affecting the accuracy.
Characterization of antral gastrin cells with region-specific antisera.
Authors: Larsson L I , Rehfeld J F
Published: April 2011

A number of gastrin antisera, which in radioimmunoassay systems showed no or negligible cross-reactivity towards the structurally and functionally related peptide cholecystokinin were found to react with both gastrin and cholecystokinin cells when used for immunocytochemistry. This discrepancy was shown to be due either to reactivity against a COOH-terminal region common to gastrin and cholecystokinin or to the occurrence of heterogenous antibody populations in the antisera. By differential absorptions the latter type of antisera could be rendered specific for gastrin. Antisera reactive against the NH2-terminal, middle or COOH-terminal regions of human heptadecapeptide gastrin were prepared and together with a specific cholecystokinin antiserum used for the characterization of antral gastrin cells of different species. The results indicate that only the COOH-terminal region of gastrin is conserved during evolution.
Electron microscope immunohistochemical localization of substance P in the central nervous system of the rat.
Authors: Pelletier G , Leclerc R , Dupont A
Published: April 2011

Substance P is an undecapeptide that is distributed in many areas of the central nervous system. In order to clearly identify the structures and organelles containing substance P, an immunohistochemical localization of this peptide was performed at the ultrastructural level. Substance P was detected in dense core vesicles of many nerve endings in both the amygdaloid nucleus and the substantia gelatinosa of the spinal cord. These findings support the hypothesis that substance P could act as a neurotransmitter.
Mithramycin fluorescence for quantitative determination of deoxyribonucleic acid in single cells.
Authors: Johannisson E , Thorell B
Published: April 2011

The use of the antibiotic drug mithramycin for cytoflurometric assessment of deoxyribonucleic acid in single cells has been studied in smears of a standard cell population of rat thymocytes. The optimal staining conditions have been determined including the influence of fixation (freeze-drying, formalin and ethanol). The drug equilibration time has been estimated in relation to the concentration of the mithramycin and to Mg++ was found to enhance the fluorescence intensity produced by the mithramycin-deoxyribonucleic acid interaction. The stability and the reproducibility of the fluorescence reaction are discussed.
Ultrastructural localizations of adenosine triphosphatase activity in resting mammary gland.
Authors: Russo J , Wells P
Published: April 2011

Adenosine triphosphatase (ATPase) activity was localized at an ultrastructural level in the resting mammary glands of female BALB/c mice. A Mg++ dependent ATPase was localized in the plasma membranes of both the epithelial and myoepithelial cells of the mammary tubules. A second type of ATPase activity that was not Mg++-dependent but that was Na+ and K+ dependent was localized primarily in the plasma membranes of the myoepithelial cells. Preincubation with either ouabain or N-ethylmaleimide decreased the quantity of reaction product, indicating that both types of ATPase activity were sensitive to these inhibitors. Control media, containing adenosine triphosphate and Pb(NO3)2 without cations, demonstrated that the amount of nonezymatic hydrolysis was negligible. These differences in the cationic requirements for plasma membrane ATPase activity can be used to distinguish histochemically the epithelial from myoepithelial cells in mammary tissue.
The distribution of acid and alkaline ribonuclease activities in preneoplastic and neoplastic rat livers.
Authors: Murthy S M , Daoust R
Published: April 2011

Ribonuclease (RNase) activities revealed by the substrate film method were compared with reactions for acid and alkaline RNase obtained by lead precipitation technique in serial sections of preneoplastic livers and hepatomas. The preneoplastic parenchymal tissue giving positive reactions with ribonucleic acid films showed both acid and alkaline RNase activities by lead precipitation technique, and the area of hyperplastic nodules nonreactive against substrate films were deficient in acid and alkaline RNase activities. Preneoplastic hyperbasophilic foci and hepatoma gave weak or negative reactions by either method, but necrotic areas and stromal tissue showed appreciable RNase activities. Thus a good correlation was observed in these tissues between the RNase activities revealed by the film method and those demonstrated by lead precipitation.
5'-Nucleotidase in different populations of mouse lymphocytes.
Authors: Uusitalo R J , Karnovsky M J
Published: April 2011

The activity of 5'-nucleotidase in different populations of intact lymphocytes was studied using biochemical, cytochemical and radioautographic methods. In some strains of mice the results showed a consistent difference in 5'-nucleotidase (AMPase) content between intact thymic and splenic lymphocytes. In the R III, C 57, BALB/c, CBA and Cd-1 strains AMPase activity in the isolated splenic cells was foru to 10 times the activity of intact thymocytes. In highly enriched populations of splenic T and B cells the average AMPase activity was about the same. From separate assays it was seen that the AMPase activity in highly enriched populations of lymphoctes was variable so that within one experiment the T cells seemed to have the higher AMPase activity while in other experiments B cells shown to be more active than T cells. Ultrastructural radioautography was done to count AMPase positive cells within T and B cell populations, the latter identified b binding of I125-labelled anti-immunoglobulin. It was seen that about 50% of B cells, but only about 10% of T cells, were positive for AMPase. It is suggested that there is a subpopulation within B and T cell populations with a high membrane AMPase activity and another subpopulation with less or no enzyme activity. It is also suggested that the activity and/or the proportion of these positive cells is changing within the splenic cell population. By using cortisone to deplete the immature cells from the thymus it was seen that the remaining mature cells have about the same AMPase activity as did the immaturecells, and thus mature T cells must gain their high acitivity after leaving the thymus. By incubating splenic lymphocytes with Concanavalin A it was also seen that the immature transformed cells had the same amount of enzyme as did untransformed cells.
An improved method for the histochemical localization of glucose-6-phoshate dehydrogenase in animal and plant tissues.
Authors: Negi D S , Stephens R J
Published: April 2011

An improved procedure is reported for the histochemical localization of glucose-6-phosphate dehydrogenase in fresh-frozen sections of animal and plant tissues. Used in this procedure are an incubation medium and an inert high-molecular-weight polyvinyl alcohol as a stabilizer to check enzyme diffusion. The incubation medium consists of 10 mg each of nicotinamide adenine dinucleotide phosphate and nitroblue tetrazolium chloride and 60 mg of glucose-6-phosphate per 20 ml of 22% polyvinyl alcohol solution in 0.2 M Tris-maleate buffer, pH 7.2. Use of this medium has given consistent results with the tissues tested. Because the use of an electron-transfer agent in the incubation medium often resulted in enzyme diffusion, its use in glucose-6-phosphate dehydrogenase localization is considered undersirable. Incubation time of 15 to 30 min for animal tissue and 20 to 45 min for plant tissue of 37degreesC was found to be optimal for producing the characteristic blue formazan at the reaction sites.
Improvements of glycol methacrylate. I. Its use as an embedding medium for electron microscopic studies.
Authors: Spaur R C , Moriarty G C
Published: April 2011

The technique for using the water-soluble embedding medium glycol methacrylate has been improved for ultrastructural studies by the simplification of the method of formation of prepolymers used in embedding the tissue, by the addition of a cross-linking agent so that sections are stable in the electron beam, and by improving the softness of the blocks by the addition of a plasticizing agent. The preservation of tissue morphology has been improved by complete dehydration in glycol methacrylate monomer prior to infiltration with the prepolymer. Preservations of tissue morphology is further enhanced by complete dehydration in ethanols and embedding in the improved glycol methacrylate medium.
A simplified method of "hypothetical grain" analysis of electron microscope autoradiographs.
Authors: Blackett N M , Parry D M
Published: April 2011

An improved method for the analysis of high resolution electron microscope autoradiographs is described which is simple and quick to perform. The method uses a transparent screen superimposed over the autoradiograph prints to provide information on the distribution of grains over neighboring structures, produced by radioactive disintegrations occurring in different regions of the section. This "cross-scatter" information is used to estimate the radioactivity in different structures without the need to make any assumptions about the size, shape and arrangement of the structures within the autoradiographs being analyzed. The method allows activity in membranes and other linear structures to be determined and also the accuracy attributable to the activity values obtained.
Immunohistochemical detection of Fc receptor. I. Light microscopic demonstration of Fc receptor by using soluble immune complexes of peroxidase-antiperoxidase immunoglobulin G.
Authors: Itoh G , Miura S , Suzuki I
Published: April 2011

The mouse mesenteric lymph node cells (in the cell suspension and frozen sections) were incubated in the soluble immune complexes of peroxidase-antiperoxidase immunoglobulin G. After being washed, they were reacted with diaminobenzidine tetrahydrochloride. Light microscopically brown-colored granules were observed on the cell surface of a proportion of small lymphocytes. In frozen sections, a proportion of small lymphocytes were stained dark brown on the cell surface. Characterization and control experiments suggest that the binding of peroxidase-antiperoxidase immunoglobulin G to the cell surface is mediated by Fc receptor. Peroxidase-antiperoxidase immunoglobulin G, therefore, can be used as in indicator of Fc receptor.
Ultracytochemical demonstration of monoamine oxidase activity in nervous and non-nervous tissue of the rat.
Authors: Müller J , Da Lage C
Published: April 2011

A method is described for the ultrastructural localization of the enzyme monoamine oxidase based on the formation of an osmiophilic formazan from the tetrazolium salt (2-(2'-benzothiazolyl)-5-styrl-3-(4'-phthalhydrazidyl) tetrazolium chloride) (BSPT) and using tryptamine as substrate. It was tested in rat heart, liver, adrenal cortex and medulla, kidney, pineal gland, cerebral cortex and cerebellar cortex and was found to provide good ultrastructural preservation, high contrast of the specific, enzyme-dependent precipitate and improved sensitivity. Monoamine oxidase activity was found in the mitochondrial outer compartment, nuclear envelope and endoplasmic reticulum in all organs studied; it was also seen occasionally in plasma membranes of liver and heart. Similar localizations were observed in interstitial cells and capillary endothelial cells. The enzyme-dependent nature of the observed precipitate was verified by control experiments consisting of incubation of overfixed tissue, omission of substrate from the incubation medium, and incubation in the presence of a specific, nonreversible inhibitor, pargyline. These studies confirm and extend the results of cell fractionation studies using liver, demonstrate the widespread distribution of monoamine oxidase in tissues, and suggest that monoamine oxidase plays important functional roles in extraneuronal cells.