bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of primary antiserum dilution on staining of "antigenrich" tissues with the peroxidase antiperoxidase technique.
Authors: Bigbee J W , Kosek J C , Eng L F
Published: April 2011

The effect of primary antiserum dilution on staining results with the peroxidase antiperoxidase method were investigated using frozen sections of perfused rat cerebellum and optic nerve. Results comparable to formalin fixed and paraffin embedded tissue were attainable only when low antiserum concentrations were used. Optimal staining of antigen rich tissue, such as frozen sections, with the peroxidase antiperoxidase method required low antiserum concentrations apparently to minimize the binding of both antigen-binding fragments of the bridging antibody to the tissue bound antiserum. It appears that low antiserum concentration insures that sufficient bridge antibody molecules will be only singly bound and thus free to attach the peroxidase antiperoxidase complex.
Bone marrow cell scene segmentation by computer-aided color cytophotometry.
Authors: Aus H M , Rüter A , Meulen V T , Gunzer U , Nürnberger R
Published: April 2011

Computer scene segmentation of touching cell images in bone marrow, on the basis of color information, is achieved using digitized scans at three different wavelengths of light. With trivariate histograms and Euler's coordinate transformation, it is possible cytophotometrically to isolate, on the basis of chromatic differences, individual heterogeneous cells located in cell groups. The ability of the described computer methods to isolate correctly the touching cell images is determined by visual comparison of the cells as seen in the microscope and the computer-generated displays of the scanned and segmented scenes.
Estimating false positive and false negative error rates in cervical cell classification.
Authors: Oliver L H , Poulsen R S , Toussaint G T
Published: April 2011

The performance of a cell recognition system on unknown data is often estimated in terms of its error rates on a test set. This paper investigates methods for producing estimates of error rates in cervical cell classification. Classification performance curves calculated using these methods are given for several classification schemes used to classify 1500 cervical cells.
Computer-assisted karyotyping with human interaction.
Authors: Oosterlinck A , Van Daele J , De Boer J , Dom F , Reynaerts A , Van Den Berghe H
Published: April 2011

A system for machine assisted karyotyping and chromosome analysis has been developed. The system uses a drum- or TV-scanner as input device, runs provisionally in 32 K memory, and also allows human interaction on several stages. The accuracy with which banded chromosomes are karyotyped depends strongly on the type of classifier and varies from 40 up to 80%. The accuracy of the human assisted classifier (98%) comes close to that of a skilled technician (99.5%) using manual chromosomal analysis. Due to technical and memory limitations, the time necessary for the karyotyping of one cell is too long and depends on the interaction time; however karyotyping within 5 min, including human interaction, will be possible in the near future.
Hydrodynamic orientation of cells.
Authors: Fulwyler M J
Published: April 2011

Chicken erythrocytes orient when caught in the confluence of two sheath-liquid streams. Preliminary results indicate that this phenomena might be used to align cells for morphologic analysis in flow.
Cytomat-R: a computer-controlled multiple laser source multiparameter flow cytophotometer system.
Authors: Shapiro H M , Schildkraut E R , Curbelo R , Turner R B , Webb R H , Brown D C , Block M J
Published: April 2011

A multiple illumination wavelength multiparameter flow cytophotometer system, using laser sources and controlled by a small, general-purpose digital computer, has been produced for use in the development of new flow cytometric techniques. Three different laser wave-lengths can be used simultaneously to illuminate different regions of the flow chamber; as many as five measurements of light scattering at various angles, extinction, and fluorescence at one or more wavelengths can be made at each illuminated station. Cells in suspension may be examined at rates of 1000 cells/sec, with seven correlated optical measurements being recorded for each cell. A library of programs for data manipulation and statistical analysis make it possible to use the system to develop and implement cell characterization, counting and classification procedures for basic and clinical research applications.
Toward organism identification through analysis of bacterial colonies.
Authors: Graham M D
Published: April 2011

The clinical problem of bacterial identification has been approached by applying pattern-recognition techniques to multi-wavelength surface-scattering and reflectance data derived from real-time scans of isolated colonies. Preliminary results, obtained from blood-agar plates inoculated with a mixture of staphylococci, streptococci and escherichieae, indicate that these organisms can be differentiated with better than 90% certainty, provided the colonies are physically separated and their growth conditions closely controlled. Data collection and classification characteristics of the experimental system are briefly described; it is felt that the technique, possibly expanded to include boundary characteristics of the colonies, may offer a viable means of identifying clinically important bacteria.
Analysis and sorting of living cells according to deoxyribonucleic acid content.
Authors: Arndt-Jovin D J , Jovin T M
Published: April 2011

The methods for measuring the deoxyribonucleic acid content of individual mammalian cells and sorting them on the basis of this parameter have until now required fixation or other treatment which renders the cells nonviable. Using a class of bis-benzimidazole dyes, Hoechst 33258 and 33342 and a multiparameter computer-controlled cell sorter, we have been able to stain and separate living cells in the G1, S, and G2+M phases of the cell cycle and to continue their growth in tissue culture with high retention of viability (greater than 90%) and no increase in heteroploidy. The quenching of the fluorescence of the bound dye by 5-bromodeoxyuridine incorporated into cellular deoxyribonucleic acid is being used with the flow system to detect and isolate mutants in deoxyribonucleic acid metabolism spectroscopically.
Flow system fluorescence polarization measurements on fluorescein diacetate-stained EL4 cells.
Authors: Epstein M , Norman A , Pinkel D , Udkoff R
Published: April 2011

We have adapted a multiparameter cell sorter to measure the distribution of fluorescence polarization in cell populations. Measurements carried out on EL4 cells show that the percent polarization of fluorescein fluorescence decreases with increasing fluorescence intensity. This inverse relationship between polarization and intensity is shown both within the cell population and by the average values of the two quantities during both the increase and decrease of fluorescence intensity. The quantitative relation between intensity and polarization is different in hypertonic than in isotonic media. These results suggest that polarization measurements carried out at a fixed time after incubation of cells with fluorescein diacetate, which is converted to fluorescein within the cells, may depend in part on the rate of fluorescein accumulation, and that agents that have been reported to change the polarization of fluorescein in living cells may do so by changing the kinetics of fluorescein accumulation.
Ultrasonic disaggregation of cell clusters.
Authors: Garcia G L , Tolles W E
Published: April 2011

A technique of cluster disaggregation has been developed and applied to cervical cells in suspension. Preliminary results indicate that ultrasound is capable of cluster disaggregation under controlled energy conditions. Cytologic quality of the resulting preparation is adequate for cell analysis in flow systems.
A procedure for dissociating Ayre scrape samples.
Authors: Leif R C , Nordqvist S , Clay S , Cayer M , Ingram D , Cameron B F , Bobbitt D , Gaddis R , Leif S B , Cabanas A
Published: April 2011

The dissociation of cervical cell suspensions after various chemical and enzymatic treatments was monitored by using the Centrifugal Cytology rotor to produce glutaraldehyde-fixed dispersions on conventional microscope slides and subsequent Pap staining. A special program was written in RPG II to record and analyze the results of the dissociation experiments in terms of white blood cells and the true cervical cells ("other cells"), and the degree of dissociation and recovery of both classes of cells. Since accurate differential counts on the untreated Ayre scrapes were difficult, the samples were syringed gently to break up the large or adventitious clumps. Cumulated results from control preparations indicate that the white blood cells and "other cells" are composed respectively of 92 and 63% single cells. The cells were further dissociated by: dissolving the cervical mucin sequentially with dithiothreitol and iodoacetic acid; depolymerizing the nucleohistone gel with ribonuclease; solubilizing the desmosomes with EDTA; removing the remaining cellular agglutinins with Varidase; and finally mechanical dispersion by hypertonic shock. The optimum procedure for dissociation involves the use of ribonuclease, dithiothreitol, iodoacetic acid EDTA, Varidase and sucrose shock. The white blood cells are now monodisperse and 81% of the "other cells" are found as single cells. If nuclear separation by two diameters is considered sufficient 98% of the "other cells" are single. The slide preparations are now sufficiently good that a scanning system is feasible.
Crypt cell isolation in the small intestine of the mouse.
Authors: Kremski V C , Varani L , DeSaive C , Miller P , Nicolini C
Published: April 2011

A successful method has been developed for isolating viable single cell suspensions of intestinal crypt cells from the small bowel of the mouse. The lumen of the intestine was perfused with a 0.2% trypsin solution that dissociated the lining epithelial cells. Crypt cell isolation, which proves to be extremely critical, occurred under optimal mechanical and chemical configurations about 75 min after the beginning of the procedure.
The unlabeled antibody enzyme method. Attempted use of peroxidase-conjugated antigen as the third layer in the technique.
Authors: Sternberger L A , Petrali J P
Published: April 2011

The use of covalently bound peroxidase-immunoglobulin conjugate has been compared with soluble peroxidase-antiperoxidase complex as antigen in the third layer of the unlabeled antibody enzyme method. The conjugate conferred no staining. The low sensitivity of the conjugate appeared to be due in part to interference by unconjugated immunoglobulin and in part to diminution of antigenic reactivity of immunoglobulin as a result of conjugation. The triple specificity amplification inherent in the unlabeled antibody enzyme method with peroxidase-antiperoxidase complex may be lost if a peroxidase-immunoglobulin conjugate could be prepared in such a manner that staining occurs.
The quantitative histologic distribution of pyridoxal phosphate in the rat stomach, effects of drugs influencing secretion.
Authors: Von Redlich D , Glick D , Bixby J A , Lees C D
Published: April 2011

The quantitative histologic distribution of pyridoxal phosphate (PLP), the coenzyme of histidine decarboxylase in the biosynthesis of histamine as well as the coenzyme of other enzyme systems, was established in the rat glandular stomach in different secretory states. This was accomplished by analyzing PLP with an improved fluorometric microprocedure in histologically identified fresh-frozen microtome tissue sections cut serially through the stomach wall of fed and 24-hr fasted rats, and of the latter injected with drugs that stimulate (histamine, theophylline and urecholine) or inhibit (atropine) secretion. In fasted compared with fed rats, and in fasted rats injected with histamine (low dose) or theophylline, compared with saline injected controls, the PLP level was reduced not only in the chief cell region where PLP is utilized in histamine production, but also in the parietal and mucous neck cell region, indicating PLP function in other enzyme systems in these sites. No change in PLP level occurred when histamine was injected at a high dose known to suppress histidine decarboxylase activity. Administration of urecholine or atropine had negligible effect on the PLP levels, indicating a lack of vagal stimulatory or inhibitory influence.
The two dimensional structure of tetrazolium-mucopolysaccharide complexes deduced from reactivity studies.
Authors: DeBari V A , Needle M A
Published: April 2011

The formation of complexes between mucopolysaccharides and tetrazolium salts has been studied both by turbidimetry and nephelometry. The technique of laser nephelometry allows the detection of colloidal aggregates in systems which may, by turbidimetric methods, be ambiguous. The results indicate that binding of tetrazolium salts to polyanions can result in soluble as well as insoluble complexes; the monotetrazolium salts form soluble complexes and the ditetrazolium compounds form insoluble complexes. The insoluble complexes are stable at relatively low pH, and are disrupted during reduction to the formazan. Complex formation is decreased at high ratios of heparin to tetrazolium, and divalent cations, even at high concentration, do not precipitate mucopolysaccharides. It is concluded that stable ionic interactions with spatial charge separation are responsible for the cross linking of the mucopolysaccharides and the formaiton of large insoluble aggregates.
Cytofluorometric quantitation of 5-hydroxytryptamine and heparin in individual mast cell granules.
Authors: Gustafsson B , Enerbäck L
Published: April 2011

Cytofluorometric quantitation of 5-hydroxytryptamine (5-HT) and heparin in individual mast cell granules is described. The technique is based on micromanipulation of intact mast cells reacted with formaldehyde or stained with Berberine sulfate and the use of a cytofluorometer equipped with a sensitive peak detecting device. The quantities of 5-HT and heparin contained in mast cell granules which are of the order of 10(-16) and 10(-13) g, respectively were expressed as relative fluorescence guanta. The results of measurements on representative samples of mast cell granules indicate that all granules contain heparin as well as 5-HT, and that there are large variations in both 5-HT and heparin content within the granule populations of individual cells. A dose dependent increase in 5-HT content in both cells and individual mast cell granules occurred 24 hr after the injection of 10--50 mg L-5-hydroxytryptophan/kg intraperitoneally. There was no evidence for an increase in the heparin content of granules or cells, indicating that a new synthesis of granular macromolecules is not required for the 5-HT uptake. The results further suggest that 5-HT may be stored initially in a cytoplasmic extragranular pool and then taken up in the mast cell granules.
Tannic acid-metal salt sequences for light and electron microscopic localization of complex carbohydrates.
Authors: Sannes P L , Katsuyama T , Spicer S S
Published: April 2011

Use of tannic acid (TA), in sequence with ferric chloride, uranyl acetate or gold chloride resulted in staining of selective but sometimes different sites in paraffin sections. TA-uranyl acetate of TA-ferric chloride stained sites rich in complex carbohydrates, wherease TA-gold chloride stained the collagen of various connective tissues different shades of red-purple to gray-black. Applied to epoxy-embedded thin sections of tissues fixed with glutaraldehyde and not post-osmicated, TA-uranyl acetate and TA-ferric chloride imparted density to subcellular sites known to contain a high concentration of mucosubstances, such as secretory granules and cisternae of the Golgi complex of certain cells. TA-gold chloride proved unsatisfactory for ultracytochemistry because of its tendency to form globular precipitates on thin sections. The effect of blockage procedures at the light microscopic level indicated that vicinal glycols are not required for binding of TA to tissue sites. Electrostatic forces were shown to be of minimal significance, whereas hydrogen bonding appeared to play a part in both TA-tissue and TA-metal binding mechanisms.
Histochemical and cytochemical localization of (Na+-K+)-activated adenosine triphosphatase in the acini of dog submandibular glands.
Authors: Nakagaki I , Goto T , Sasaki S , Imai Y
Published: April 2011

p-Nitrophenyl phosphatase (p-NPPase) activity of (Na+-K+)-activated adenosine triphosphatase ((Na+-K+)-ATPase) on the acinar cells of dog submandibular gland was demonstrated by using light microscopy. The reaction products of p-NPPase of fresh frozen sections were seen to be localized on the basal parts of acini, and disappeared when the sections were incubated in medium containing 10(-3) Mouabain or in a K-free medium. Under the electron microscope, the reaction products of ATPase were found to be localized on the basolateral plasma membrane of both serous and mucous cells. On the microvilli of the luminal plasma membrane of the acinar cell, a small quantity of the reaction products was also present. This localization of ATPase reaction products on the serous and mucous cells seemed to coincide well with that of p-NPPase activity observed on the acini under light microscopy. Possible explanations are given regarding distribution of the above mentioned enzymes in relation to the cation transport of the plasma membrane. Structural and functional asymmetrical properties of acinar cells of the dog submandibular gland are also discussed.
Visualization of intracellular concanavalin A binding sites in retinal photoreceptors.
Authors: Nir I
Published: April 2011

Localization of carbohydrate components in retinal photoreceptor cells and membranes was studied. Frog and rat retinas were fixed with glutaraldehyde and embedded in glycol methacrylate or in a mixture of glycol methacrylate, glutaraldehyde and urea. Thin sections were incubated with ferritin-labeled concanavalin A (F-Con A) and stained with osmium vapors. Intensive binding was observed in both rod and cone outer segments. In the rod inner segment, differential binding of F-Con A was demonstrated. While numerous ferritin granules were observed in the myoid zone, only a few were seen in the ellipsoid zone, except for a local accumulation along the plasma membrane. In the rod outer segment, Con A binding sites were closely associated with the disk membranes. Ferritin granules were observed on both sides of the membranes. The relationship between the localization of Con A binding sites and the orientation of visual pigment molecules within the rod outer segments disk membranes was discussed.
Immunocytochemical localization of renin in the submandibular gland of the mouse.
Authors: Gresik E , Michelakis A , Barka T , Ross T
Published: April 2011

Renin was localized in the submandibular gland of the adult mouse at light and electron microscopic levels by the unlabeled antibody enzyme method of Sternberger. At the light microscopic level, renin was confined to the granular convoluted tubule (GCT) segment of the gland with considerable variation among GCT cells in intensity of staining. Some GCT cells failed to stain for renin. The pattern of staining was the same in the gland of male and female mice, but in the glands of females GCT segments were smaller and less numerous. At the electron microscopic level, staining for renin was also confined to the GCT cells, and was localized exclusively to the secretory granules. The intensity of staining of the secretory granules within a given GCT cell varied; some cells contained only minimally reactive or negative secretory granules. All other organelles within the GCT cell, except condensing vacuoles, failed to stain.
Developmental distribution of microperoxisomes in the rat submandibular gland.
Authors: Mooradian B A , Cutler L S
Published: April 2011

The present study investigated the size, number, and distribution of microperoxisomes (MP) during the prenatal and postnatal development of the rat submandibular gland (SMG). A three-fold increase in MP number per cell was observed in the cells of the rudiment from the 15th to the 16th day of gestation. The early secretory and striated duct cells contained about 9.0 MP. The number of MP per secretory cell decreased such that 3.5 MP were found in each mature acinar cell. In the striated duct cells, MP number progressively increased to 40.0. As the convoluted granular tubule cells (CGT) developed from striated duct cells there was an increase in MP number from 16.0 to 26.0/cell. At maturity, the convoluted granular tubule cells contained only 14.0 MP. Throughout development of the SMG, intercalated duct cells showed only rare MP. The data suggests that the number, size, and distribution of MP changes as a function of the particular path of differentiation followed by the various cells in the rat SMG.
Immunoperoxidase stains cortisol in adrenal and pituitary.
Authors: Dornhorst A , Gann D S
Published: April 2011

The unlabeled peroxidase-anti-peroxidase (PAP) method of Sternberger was used to localize cortisol within paraffin embedded sections of cat adrenal and pituitary tissue. Incubation of the cortisol antiserum used in this method with increasing concentrations of cortisol led to progressive extinction of cortisol staining of the adrenal fasciculata cells, (as measured with a scanning integrating microdensitometer). This result suggests strongly that the staining achieved with this method was specific for cortisol. Cortisol staining was demonstrated not only within cells that synthesize cortisol (the adrenal fasciculata) but also in cells of the adrenal medulla and of the anterior pituitary, two target sites for cortisol action.
Ultrastructural localization of pancreatic polypeptide in the F cell of the dog pancreas.
Authors: Greider M H , Gersell D J , Gingerich R L
Published: April 2011

The F cell of the dog pancreas has been identified as the specific cell type containing pancreatic polypeptide. This localization of pnacreatic polypeptide was accomplished by immunocytochemical staining of ultrathin sections and direct electron microscopic identification. Verification of the specificity of the reaction was obtained by blocking experiments on serial sections of the same cell. It is proposed that the name F cell be used for defining in all species the islet cell that contains pancreatic polypeptide.
Microfluorimetric scanning of sympathetic nerve fibers. An improved method to quantitate formaldehyde induced fluorescence of biogenic amines.
Authors: Schipper J , Tilders F J , Ploem J S
Published: April 2011

A two dimensional scanning procedure was developed for studying quantitative aspects of formaldehyde induced fluorescence (FIF) from noradrenaline containing nerve fibers. A computer (PDP 12) controlled scanning device was equipped with a cooled photomultiplier, a 0.5 micron scanning stage and a 0.5 micron measuring spot. Photodecomposition was reduced by a high scanspeed (100/sec) and a small excitation field. Digitized images were obtained with a high resolution. Data (10,000/scan) of large scan areas (2500 micron2) in the iris of the rat were transformed into fluorescence histograms, which allowed statistical evaluation. Visible changes induced in the histochemical preparation by administration of drugs (reserpine, L-Dopa) were reflected in characteristic changes in the histograms. A minor reduction in noradrenaline content, which was too small to be detected by visual observation of the microscopical image and by classical microfluorimetric methods, did evoke a significant change in the histograms. It is concluded that microfluorimetric scanning is a very sensitive technique for detecting changes of formaldehyde induced fluorescence from neuronal networks.
A correlative chemical and histochemical study of the O-acetylated sialic acids of human colonic epithelial glycoproteins in formalin fixed paraffin embedded tissues.
Authors: Reid P E , Culling C F , Dunn W L , Clay M G , Ramey C W
Published: April 2011

Procedures are described for the isolation and identification of epithelial glycoproteins from formalin fixed, paraffin embedded specimens of human large intestine. The side chain O-acetylation patterns of the sialic acids of these glycoproteins were surprisingly similar to those of purified glycoproteins prepared from epithelial cells obtained from the same tissue before fixation. These results were consistent with those obtained by histochemical procedures performed on representative sections taken from the same tissue blocks. The methodology described permits a direct correlation of chemical and histochemical results obtained from the study of colonic epithelial glycoproteins of both normal and diseased tissues. It eliminates some of the difficulties associated with interpretation of the results by either discipline and may provide new information which would be unavailable by either chemistry or histochemistry alone.
Feulgen-DNA absorption spectrum of euchromatin and constitutive heterochromatin.
Authors: Mello M L
Published: April 2011

When the heterochromatin and euchromatin of Triatoma infestans are maximally depurinated, their Feulgen-DNA spectral profiles exhibit a shoulder at about lambda = 530 nm. The shoulder is especially prominent in the curves of the heterochromatin. It is assumed that this pattern is related to richness in repetitive DNA in the heterochromatin, favoring appearance of Schiff reagent molecules di-substituted with adjacent apurinic acid aldehydes.
Immunochemical characterization and cellular localization of pepsinogens in cat and dog.
Authors: Liebman W M , Samloff I M
Published: April 2011

The antigenic relationships and cellular localization of cat and dog pepsinogens were investigated by electrophoretic analysis, immunodiffusion, immunoelectrophoresis, immunoabsorption, and by immunofluorescence, respectively. Rabbit antiserum to human and hog group I (Pg I) and group II pepsinogens (Pg II) had been previously prepared. Electrophoretic analysis revealed at least eight distinct proteases in extracts of gastric and proximal duodenal mucosa, resistant to alkalinization but destroyed by sequential accidification and neutralization. Rabbit antiserum to Pg I (anti-Pg I) and Pg II (anti-Pg II) produced a single precipitin arc against each extract forming a line of nonidentity. Immunoelectrophoresis of extracts produced a single precipitin arc against anti-Pg I or anti-Pg II. The specificity of the antibodies for the group I or group II pepsinogens was confirmed by immunoabsorption. By immunofluorescnece, both Pg I and Pg II were present in mucous neck and chief cells in fundic mucosa, in the pyloric gland cells in antral mucosa, and Brunner's glands in the proximal duodenum. The results indicate that canine and feline pepsinogens are electrophoretically heterogenous, that canine and feline Pg I share antigenic determinants with each other but not with Pg II, that a similar positive relationship exists for Pg II, and that both Pg I and Pg II are localized to the peptic cell mass, consisting of four types of cells.
Adenylate kinase activity in various organs and tissues of mice with the obese-hyperglycemic syndrome (gene symbol Ob/Ob).
Authors: Borglund E , Brolin S E , Agren A
Published: April 2011

The activities of adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) in lyophilized cryostat sections of various organs from mice with the obese-hyperglycemic syndrome (gene symbol ob/ob) were measured fluorometrically. Skeletal and heart muscle tissues had the highest enzyme activities, i.e. 258 and 155 mmoles substrate converted per kg dry weight and per hr (MKH), respectively. Pancreatic islet specimens, which were composed mainly of B-cells, possessed the highest activitty of all parenchymatous cells studied, with 18 MKH as compared to 16 MKH for the cortical tubules of the kidney and 7 and 6 for the exocrine pancreas and liver cells, respectively. The granular layer of the cerebellum had enzyme activity of 31 MKH. The adenylate kinase activity in the B-cells was apparently not influenced by hyperglycemia since it was similar both in obese-hyperglycemic mice of different ages and in lean mice, although the blood sugar concentrations in such animals are much different.
Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents.
Authors: Mesulam M M
Published: April 2011

Tetramethyl benzidine (TMB) is a presumptively non-carcinogenic chromogen which yields a blue reaction-product at sites of horseradish peroxidase activity. Sixty-six distinct procedures were performed in rats and monkeys in order to determine the optimal incubation parameters for TMB. As a result, a procedure is recommended whose sensitivity greatly surpasses that of a previously described benzidine dihydrochloride method. Indeed, the sensitivity of this new method in demonstrating retrograde transport is markedly superior to that of the previously described benzidine dihydrochloride method. Furthermore, as a consequence of this enhanced sensitivity, many efferent connections of the injection site are also visualized. The injection site demonstrated by this TMB procedure is significantly larger than the one demonstrated when benzidine dihydrochloride or diaminobenzidine is used as a chromogen. Finally, this TMB procedure has been compared to two other TMB procedures and found to provide superior morphology and sensitivity.
An immunocytochemical study of a rat pituitary multipotential clone.
Authors: Bowie E P , Ishikawa H , Shiino M , Rennels E G
Published: April 2011

The 2A8 clone, a normal diploid rat anterior pituitary cell strain, was investigated by immunocytochemistry to determine the cell types into which the clonal cells differentiated in vivo and in vitro. The in vivo study was carried out by injecting the 2A8 clone preparation either into the hypothalamic region or under the kidney capsule. After thirty days the implants were removed and studied by immunocytochemistry. In vitro, many prolactin cells and a few growth hormone cells were found. In vivo, however, prolactin, growth hormone, ACTH and TSH cells and gonadotrophs were identified. We concluded that the 2A8 clone was multipotential. Since the gonadotrophs of the implants made in the hypothalamic region were larger and more plentiful than those in the kidney implants, and since gonadotrophs were lacking in the in vitro system, it appeared that the hypophysiotrophic environment was the most conducive to gonadotrophic differentiation and maintenance, and that the factor or factors necessary for cyto-differentiation were apparently present in the general circulation of the rat but absent in the growth medium of our culture cells.
A simple procedure for distinguishing dopamine from noradrenaline in peripheral nervous structures in the fluorescence microscope.
Authors: Hess A
Published: April 2011

The difference between dopamine and noradrenaline after ordinary histofluorescent procedures cannot be discerned. Reserpine treatment results in depletion of fluorescent material from dopaminergic and noradrenergic peripheral nervous structures. Administration of reserpine, 1 mg/kg subcutaneously for 3 hr, followed by intraperitoneal injection of 200 mg/kg levodopa methyl ester on 0.9% saline for 90 min, result in refluorescence of dopaminergic (glomus cells of the carotid body) but not noradrenergic (sympathetic ganglion cells, nerves of atrial heart muscle and blood vessels) structures. Hence, the sequential administration of these readily available drugs and the application of ordinary histofluorescent techniques result in a simple procedure for distinguishing dopamine from noradrenaline in the fluorescence microscope.
Microspectrophotometric quantitation of the diaminobenzidine reaction for histochemical demonstration of cytochrome oxidase activity.
Authors: Frasch A C , Itoiz M E , Cabrini R L
Published: April 2011

Polyacrylamide models in which an extract of cattle heart mitochondria was incorporated, as well as cryostat sections of tongue muscle and epithelium, were used to set up the conditions under which the histochemical reaction for the demonstration of cytochrome oxidase can be quantitated. Using diaminobenzidine in a concentration of 5.5 mM, cytochrome C in a fixed concentration of 76 micron and keeping the incubation medium away from direct light action, enzyme activity can be evaluated by means of direct microphotometry on tissue sections. Each biologic model requires previous individual determination of the measurement limits. These limits can be readily established by using a small chamber for the incubation medium, which can be placed in the microphotometer, allowing the reaction rate to be following using a single section.
The use of the fluorescent probe 8-anilinonaphthalene sulfate (ANS) for collagen and elastin histochemistry.
Authors: De Campos Vidal B
Published: April 2011

The reactivity of skin elastin and collagen to ANS (8-anilinonaphthalene-1-sulphonate) dissolved in nonpolar (e.g. butanol) and polar (distilled water, phosphate buffer at pH 2.8 and 8.2) solvents was compared in histological sections. The elastin fibers were demonstrated to differ from collagen at the fluorescence microscopy level under certain ANS staining conditions. When treated with butanol-ANS solutions, both fiber types exhibit a blue fluroescence with spectral emission profiles alike. However, the elastin emission values in fluorescence-relative units are larger than those exhibited by collagen. On the other hand, for fibers treated with ANS dissolved in polar solvents, elastin and collagen differ in terms of wavelengths of their emission maxima. The ANS attachment to substrate is ascribed to be predominantly nonpolar for dye butanol solutions. When the dye is dissolved in polar solvents, however, polar bindings between ANS and the stained substrate may be established that contribute to emission maximum shift to longer wavelengths.
Flow-cytometric analysis of chicken red blood cells.
Authors: Bloch D , Beaty N , Fu C T , Chin E , Smith J , Pipkin J L
Published: April 2011

Flow-cytometric analysis of acriflavin-Feulgen stained chicken erythrocytes shows a complex distribution of amounts of deoxyribonucleic acid fluorescence, the profile consisting of a main peak and a right hand shoulder. This bimodal distribution, an artifact characteristically seen on analysis of flattened cells using orthogonal flow systems, results from fluorescence emission in preferred directions stemming from the combined effects of refractility and orientation of the cells. The shoulder disappears on analysis of lysed erythrocyte ghosts, also on analysis of cells in a medium whose refractive index approximates that the cells. An orientation effect for matrue erythrocytes was indicated by reanalysis of fractions after sorting on the basis of high and low fluorescence or scatter signals. Both fractions gave the original range of values on reanalysis, although some changes in shape of the profile and in the peak positions for the sorted cells were seen. Sodium dodecyl sulfate treatment of stained cells "loosened" the cells' structure, yielding lowered scatter values, and fluorescence values approaching those of the shoulder. The average fluorescence emission of the erythrocytes was lower than that of reticulocytes and lymphocytes. The values of the latter correspond closely, although coincidently, to that the erythrocyte shoulder values. Dual parameter analysis of forward light scatter, and fluorescence, which was detected at 90 degrees to the laser beam, showed the low fluorescence to be accompanied by low scatter signal, and the high fluorescence among the cells with the high scatter signal. The lowered forward scatter signal is due to a wider scattering of light from cells oriented edge-on to the detector, and loss of signal beyond the acceptance angle of the detector. These results suggest that the preferred directions for fluorescence are in the plane of the cells, and the values are dependent on the cells' orientation in the stream. These interpretations were supported by the results of analysis of partially oriented cells. The approaches used and conclusions arrived at are similar to those of Gledhill et al (16), Van Dilla et al (37), in their analysis of fluorescence of flat sperm cells although the affects in the case of the erythrocytes are less extreme.
A detection method for lactic dehydrogenase activity in the inner ear.
Authors: Omata T , Ohtani I , Ohtsuki K , Ouchi J
Published: April 2011

A method for the detection of lactic dehydrogenase enzymatic activity in outer hair cells of the rabbit is described. The membranous labyrinth with temporal bone was prefixed in glutaraldehyde. After being placed into the incubation medium, it was postfixed in osmium tetroxide. Specimens of the organ of Corti were removed. Then the specimens were embedded in water-soluble glycol and cut with a cryostat for light microscopy, and also they were embedded in Epon and cut for light and electron microscopy. Sectioning of the membranous labyrinth was very easily made when the specimens were embedded in both the water-soluble glycol and the Epon. The structures of the frozen sections as well as the Epon-embedded ones were well preserved. In the frozen sections the preservation and localization of reaction products were thoroughly kept, but monoformazan of the Epon-embedded sections was soluble.
A fixation method for improved antibody penetration in electron microscopical immunoperoxidase studies.
Authors: Bohn W
Published: April 2011

A fixation method for electron microscopical immunoperoxidase staining has been developed, which (a) allows penetration of antibodies through cell membranes to intracellular antigen sites, (b) provides a reasonable cell preservation and (c) does not alter the antigenic structure in too great an extent. Penetration of the antibodies has been achieved by using saponin as a cell membrane attacking agent. The best results could be obtained after pretreatment of cell monolayers with a mixture of 0.05% saponin, 0.0125%-0.05% glutaraldehyde and 1% paraformaldehyde for 5 min at 4 degrees C, and postfixing them with the corresponding fixative without saponin for 45 min at 4 degrees C.
A new dynamic model system for the study of capture reactions for diffusable compounds in cytochemistry. I. Description of the model with special attention to phosphate capture in acid phosphatase cytochemistry.
Authors: de Jong A S , Hak T J , van Duijn P , Daems W T
Published: April 2011

A model system is described for the investigation of the dynamics of precipitation processes in a matrix. In this system a solution containing the molecular species to be precipitated and the precipitating medium are pumped along opposite sides of a polyacrylamide film. The solutions flowing continuously along the film, interact and can form a precipitate inside the film. The applicability of the model was tested on the capture reaction for phosphate ions by the Gomori type medium for acid phosphatase. Precipitation of lead phosphate in the film occurred only at a phosphate concentration above a certain value. The dependence of this minimal phosphate concentration on various parameters was studied and the results were compared with values found in earlier model studies and calculations concerning phosphate concentrations that can be built up in lysosomes during the Gomori reaction. The system seems promising for obtaining fundamental data about other cytochemical enzyme trapping reactions as well as for the matrix facotrs involved in bone calcification and shell formation.
Characterization of rat bone marrow lymphoid cells. I. A study of the distribution parameters of sedimentation velocity, volume and electrophoretic mobility.
Authors: Zeiller K , Hansen E
Published: April 2011

Various cell populations in rat bone marrow were characterized by means of a two dimensional separation using velocity sedimentation and free flow electrophoresis and by electrical sizing of the separated cells. Up to 4.5 mm/hr five different populations with discrete distributions in volume (coefficient of variation 10% to 13%) and sedimentation velocity (coefficient of variation 6% to 10%) were observed. Three of the small sized populations represented lymphocytes and small normoblasts and two of the larger sized populations represented myeloid cells. Almost all of these cells were in the G0/G1 cycle phase. In the faster sedimenting fractions which contained immature myeloid, erythroid and undefined blast cells and two S phase populations, discrete volume distributions were not evaluated. The cell populations with homogeneous volume (particularly the small lymphocytes) showed high density variations which condiserably impair the separation resolution. The cells sedimenting slower than 3.5 mm/hr were further separated by means of free flow electrophoresis into three peaks differing in electrophoretic mobility (EPM). The peaks of low and high EPM contained two populations and the peak of medium EPM contained three populations all characterized by normal volume distributions of uniform coefficient of variation between 11% and 14%. The small cells in the peaks of high and medium EPM were normolblasts and the other cells were lymphocytes. The biological significance of these results is discussed.
Ultrastructural localization of basic proteins in Trypanosoma cruzi.
Authors: Souto-Padrón T , De Souza W
Published: April 2011

The postformalin ammoniacal silver (AS) and the ethanolic phosphotungstic acid (EPTA) techniques were applied in epimastigote and trypomastigote forms of the pathogenic protozoa Trypanosoma cruzi to detect basic proteins at the ultrastructural level. With both techniques, reaction was observed in the nucleus and in some cytoplasmic vacuoles. In the kinetoplast of epimastigotes, reaction was observed only at its periphery. In trypomastigotes, however, an intense reaction was observed in the spherical kinetoplast. With the ethanolic phosphotungstic acid technique, reaction was also observed in ribosomes and at the peripheral doublet microtubules of the flagellum. The filaments which form the paraflagellar structure did not react.
Negative staining of whole cells: transmission electron microscopy of peripheral organelles in rat venous endothelial cells.
Authors: Racker D K
Published: April 2011

The study of whole negatively stained cells has revealed details of cellular organelles in rat venous endothelial cells. In particular, details of surface membrane organelles and small tubular structures were demonstrated. The surface membrane organelles which appeared "vesicular-like" were found to be connected with small tubular attachments. These findings were correlated with those described by other techniques. It is significant that this simple technique appears to permit the demonstration of fine details of three-dimensional cytoplasmic structures.
Evidence for glycogen particles in the peroxisome of riboflavin deficient mouse kidney.
Authors: Kobayashi K , Yamamoto T
Published: April 2011

In the proximal tubular cells of the kidney in riboflavin deficient mice, electron dense particulate materials of about 30 nm in diameter were observed in the matrix of the peroxisome-like cytoplasmic bodies. The cytochemical study showed that these cytoplasmic bodies were negative for acid phosphatase activity and positive for DAB reaction, thus indicating that these organelles were peroxisomes. The particulate materials in peroxisomes were well stained with aqueous uranyl acetate, lead tartrate, alkaline bismuth and periodate acid bismuth. These particles were also digested by amylase. These results indicated that the particulate materials in peroxisomes were native glycogen particles. From the present study it was apparent that the peroxisome contained glycogen particles, and suggested the possibility that in some conditions glycogen might be synthesized in the peroxisome.
Cytophotometric analyses of myocardial RNA in rats exposed to altitude hypoxia.
Authors: Stere A J , Brister N W , Anthony A
Published: April 2011

Analytical cytophotometry of azure B-stained heart sections was employed to investigate the pattern of myocardial ribonucleic acid (RNA) alterations in rats exposed to acute (1-2 days) and prolonged periods (1-8 weeks) of hypoxia exposure (380 torr). Data support the existence of a slight transient drop in myocardial RNA on day one of exposure followed by a restoration to levels slightly elevated over controls during a 1- to 8-week exposure interval. Because of the high variability in RNA levels among myocytes (coefficient of variation, ca 40%), a shift in the proportion of low and high RNA containing segments of the myocyte population proved to be a more sensitive indicator of suppression or augmentation of RNA synthesis than the use of average RNA levels of the cell population analyzed. Microscopic analyses revealed the presence of compensatory vascular responses which could be effective in ameliorating the extent of tissue hypoxemia, i.e., capillary vasodilation on day 1 with a progressive increase in vascularization with prolonged exposure.
The effect of aldehyde fixation on selected substrates for energy metabolism and amino acids in mouse brain.
Authors: Conger K A , Garcia J H , Lossinsky A S , Kauffman F C
Published: April 2011

The effect of aldehyde fixation on concentrations of low molecular weight constituents was determined by comparing amounts of selected intermediates in brains of mice exposed to aldehyde fixative solutions with those perfused with phosphate buffer solution alone. Aldehyde perfusion resulted in excellent preservation of cerebral cortex ultrastructure in the presence of dramatic declines in adenosine triphosphate, phosphocreatine, glucose and glucose-6-phosphate that occureed before exposure of the tissue to aldehyde fixatives. Decreases in hexose were accompanied by approximately a 4-fold increase in lactate and a 2-fold increase in pyruvate. Glycogen levels decreased by about 60% during the initial operative procedure but remained constant after aldehyde fixation. Glycogen content declined approximately 90% in tissues that were not treated with aldehyde. Concentrations of aspartate and glutamate changed only slightly during the initial period (1-5 min) and remained constant for at least 90 min in cerebral cortices fixed with aldehydes. Alanine levels increased in both fixed and unfixed tissue; however, this increase was much smaller in tissues exposed promptly to aldehydes. Total ninhydrin-positive material in perchloric acid extracts of brain decreased in mice exposed to aldehyde solutions but increased in tissues that were not. These results indicated that several amino acids may be measured reliably in tissues preserved for light and electron microscopy. In addition, determination of glutamate: alanine ratios in tissues perfused with aldehydes may provide an indication of the timing of fixation.