bluuebunny/crossref_metadata_2025 huggingface dataset:
Biochemical and histocytochemical studies on response of ammonia-producing enzymes for nh4cl-induced acidosis.
Authors: Seyama S , Iijima S , Katunuma N
Published: April 2011

NH4Cl-induced acidosis in rats resulted in renal enlargement and increase in activities of phosphate-dependent glutaminase and glutamic dehydrogenase. The renal enlargement was associated with protein synthesis but not deoxyribonucleic acid synthesis. In control rats histochemical activity of glutamic dehydrogenase was seen dominantly in the proximal straight tubule. In acidotic rats high activity was noted in the proximal convoluted tubule as well as in the proximal straight tubule. By electron microscopy reaction product was in mitochondria. The results suggest that urine ammonia is produced in mitochondria of epithelial cells in the proximal straight tubule in both normal and acidotic rats. Increased enzyme activity in acidotic rats is largely associated with epithelial cells of the proximal convoluted tubule.
The dissociation of transplantable tumors.
Authors: Noel J S , Zucker R M , Wu N C , Demaray S Y
Published: April 2011

Four animal transplantable solid tumors, composed of varying morphologic architecture and intercellular specializations, were studied by light and electron microscopy. These tumors were dissociated into viable single cell populations using a combination of mechanical and enzymatic methods. The conditions necessary for optimal dissociation consisted of (a) preparation of the tumor to maximize the tissue surface area, (b) enzymatic digestion with continuous agitation and (c) additional agitation to release loosely attached cells. Other factors that influenced the dissociation were optimized and discussed.
Laser cytophotometric detection of variations in spatial distribution of concanavalin-A cell surface receptors following viral infection.
Authors: Leary J F , Todd P
Published: April 2011

Human cells in culture (H.Ep. 2) were infected with herpes simplex virus type 2 at a multiplicity of infection (m.o.i.) of 3 for 3, 18 and 36 hr. Spatial redistribution of concanavalin (Con-A) cell surface receptor sites was detected by an indirect double-antibody (peroxidase-conjugated goat anti-rabbit to rabbit anti-Con-A) immunoenzymatic staining reaction which rendered the Con-A sites visible to light microscopy and to flow laser cytophotometry. Significant spatial redistribution of cell surface Con-A receptor sites occurred within the first 3 hr after infection with herpes simplex virus type 2. The infected cells must be in the presence of Con-A for the redistribution to occur, and significant redistribution occurs only at 37 degrees C. Fixation of infected cells prior to application of Con-A prevented this spatial redistribution of Con-A receptor sites.
Quantitative extraction of morphologic cell parameters from the diffraction pattern.
Authors: Seger G , Achatz M , Heinze W , Sinsel F
Published: April 2011

A new technique for the fast and quantitative extraction of cell parameters from diffraction patterns was applied to cervical gynecologic material. Based on radial scans through the diffraction pattern, this technique permits the simultaneous determination of the nuclear and cytoplasmic diameter by Fourier analysis of the radial scanning signals after compensation for the intensity falloff by an amplification which is proportional to the third power of the radial position in the diffraction plane. In addition, a correlation between the angle of the highest amplitude of the scanning signals and the coarseness of microstructures in the cell was observed. Illustrative examples of measurements on exfoliated cells of different type are presented.
Uniform lateral orientation, caused by flow forces, of flat particles in flow-through systems.
Authors: Kachel V , Kordwig E , Glossner E
Published: April 2011

Recently, it was shown that the lateral orientation of sperm cells disturbs the deoxyribonucleic acid distribution measured by fluorescence in a laterally laser-illuminated flow system. The present results show how flat particles may be influenced to assume a uniform lateral orientation. This was achieved by choosing the geometrical dimensions of the hydrodynamic focusing flow path. High speed photographs of fixed chicken erythrocytes oriented in experimental chambers are presented.
A high efficiency flow cytometer.
Authors: Skogen-Hagenson M J , Salzman G C , Mullaney P F , Brockman W H
Published: April 2011

A flow chamber has been developed which collects about 60% of the total cell fluorescence for analysis compared to about 2.5% for conventional flow systems. The chamber, an ellipsoid of revolution, is gold-plated for increased reflectivity. Fluorochrome-stained cells enter the flow cell directly above the primary focus of the ellipsoid at the rate of 1000 cell/sec. A focused argon-ion laser beam enters the flow cell parallel to the semiminor axis and intersects the cell stream at the primary focus. Fluorescent light emanating from this point is reflected toward the secondary focus, where it exits the chamber for analysis. The high efficiency flow cytometer has been used to obtain nucleotide fluorescence distributions from samples of Micrococcus glutamicus bacteria stained with propidium iodide and of spermatozoa stained by the acriflavine-Feulgen procedure.
Identification of cell asymmetry and orientation by light scattering.
Authors: Loken M R , Parks D R , Herzenberg L A
Published: April 2011

Light scattering from chicken red blood cells has been used as a model system to identify the asymmetry of cells. The histogram for forward angle light scattering for these cells is bimodal, the signal size being dependent on the cell orientation. A dual orthogonal scatter system is used to conclusively demonstrate this orientational variation in signal. A third scattering system, using a single incident beam with two orthogonal detectors, is used to further characterize the orientational variation of the scatter signal. In this third system it is shown that the signal in a detector set 90 degrees from the incident beam collects light reflected from the cell surface. The optical selection of cells in specific orientations using these systems may circumvent the need to physically orient cell in flow systems.
Fluvo-metricell, a combined cell volume and cell fluorescence analyzer.
Authors: Kachel V , Glossner E , Kordwig E , Ruhenstroth-Bauer G
Published: April 2011

A new flow through instrument that simultaneously measures cell volume (resistance pulse technique) and cell fluorescence in the same orifice will be described. The fluorescence pulses of the hydrodynamically focussed cells are picked up by the optics via the axial direction (principle of Dittrich and Goehde, Z Naturforsch 24b:360, 1969). There is no coordination problem between the fluorescence and the resistance pulses to be observed because a new type of transducer is used. The electronic system provides gating of one or two parameter histograms. Function tests are performed with the incorporated two-parameter test spectrum generator. Different examples of using the instrument in practice are shown. The volume that may be measured with an orifice of 70 micron diameter ranges between 4 and 1400 micron3 (1:350). Coefficients of variation of the fluorescence below 2% are measured.
Peculiarities of cytochemical properties of cancer cells as revealed by study of deoxyribonucleoprotein susceptibility to Feulgen hydrolysis.
Authors: Zelenin A V , Kushch A A , Chebanu T A
Published: April 2011

Chromatin of human squamous carcinoma cells reacts more intensively to short (1-2 min) acid hydrolysis in the Feulgen reaction and is, after such treatment, more intensively stained by Schiff reagent than chromatin of normal cells of the same origin. To reveal this difference in chromatin properties the use of a fluorescence variant of the Feulgen reaction is necessary because nuclei-binding of Schiff reagent after such short hydrolysis is so weak that the amount of the stain bound by means of absorption technique is hardly possible. The use of increased sensitivity of cancer cells chromatin to acid hydrolysis for cancer cytodiagnosis is suggested, especially for the diagnosis of so called diploid cancers for which detection on the basis of deoxyribonucleic acid content determination is impossible.
Virometer: an optical instrument for visual observation, measurement and classification of free viruses.
Authors: Hirschfeld T , Block M J , Mueller W
Published: April 2011

An instrument based on fluorescence correlation spectrometry and total reflection fluorescence visually and photoelectrically detects and sizes viruses at moderate concentrations in biologic fluids in minutes. Viruses can be classified using their nucleic acid type and amount determined by new fluorescent staining and data handling techniques.
Automatic measurement of sister chromatid exchange frequency.
Authors: Zack G W , Rogers W E , Latt S A
Published: April 2011

An automatic system for detecting and counting sister chromatid exchanges in human chromosomes has been developed. Metaphase chromosomes from lymphocytes which had incorporated 5-bromodeoxyuridine for two replication cycles were treated with the dye 33258 Hoechst and photodegraded so that the sister chromatids exhibited differential Giemsa staining. A computer-controlled television-microscope system was used to acquire digitized metaphase spread images by direct scanning of microscope slides. Individual objects in the images were identified by a thresholding procedure. The probability that each object was a single, separate chromosome was estimated from size and shape measurements. An analysis of the spatial relationships of the dark-chromatid regions of each object yielded a set of possible exchange locations and estimated probabilities that such locations corresponded to sister chromatid exchanges. A normalized estimate of the sister chromatid exchange frequency was obtained by summing the joint probabilities that a location contained an exchange within a single, separate chromosome over the set of chromosomes from one or more cells and dividing by the expected value of the total chromosome area analyzed. Comparison with manual scoring of exchanges showed satisfactory agreement up to levels of approximately 30 sister chromatid exchanges/cell, or slightly more than twice control levels. The processing time for this automated sister chromatid exchange detection system was comparable to that of manual scoring.
Differential fluorochromasia of human lymphocytes as measured by flow cytometry.
Authors: Wilder M E , Cram L S
Published: April 2011

Peripheral human lymphocytes reacted with fluorescein diacetate and analyzed by flow cytometry produced a bimodal fluorescence distribution that was shown to be attributable to the differential staining of T and B lymphocytes. Lymphocytes were fractionated into rosetting (T cell) and nonrosetting (B cell) populations. Both subfractions were reacted with fluorescein diacetate and analyzed by flow cytometry. The rosetting fraction was more fluorescent than the nonrosetting fraction, and the analysis of an appropriate mixture of the subfractionated populations produced a fluorescence distribution very similar to that obtained with unfractionated lymphocytes.
A cytomorphologic approach to standardization of cell dispersal evaluation for gynecologic specimens.
Authors: Barrett D L , King E B
Published: April 2011

Dispersal of cells in gynecologic specimens is a major concern in sample preparation for automated systems. A variety of dispersal techniques has been tested, with varying degrees of success reported by many laboratories. In order to have a more meaningful comparison of the results of different techniques and among investigators, a standardized evaluation procedure is desirable. The cytomorphologic element of evaluation of cell dispersal has been developed and tested in this laboratory and might be applied toward this purpose. This evaluation reveals the changes in relative numbers of single cells according to cell type and estimates cell loss as well as changes in cellular preservation. Groups of cells are tabulated according to whether they appear as definite tissue groups or as clumps of single cells. Because abnormal cells and cells from the transformation zone often occur in tissue groups, special emphasis is placed on dispersal analysis by cell type to reveal whether the diagnostically important cells and cells relating to specimen adequacy were optimally dispersed. Furthermore, the format of evaluation allows assessment of the applicability of dispersal procedures to preparation for both flow and slide-based systems of automated cytology.
Enrichment of the gynecologic cytologic specimen.
Authors: Tolles W E , Garcia G L
Published: April 2011

The principal objective of our group has been and continues to be the improvement of the biologic signal-to-noise ratio of the gynecologic cytologic specimen through: the dispersal of clusters and clumps, enrichment of the abnormal component of the dispersed specimen by the method of rate-zonal sedimentation under unit gravity, and monitoring the effects of these manipulations with biophysical non-destructive methods. This report is concerned only with presenting the current status of the effectiveness of our enrichment techniques both qualitatively and quantitatively. Zonal sedimentation creates a broad spatial spectrum of cells by virtue of their different velocities which are directly related to their areas. Certain fractions of this spectrum are extracted, concentrated and deposited on slides for subsequent manual differential analysis, evaluation and documentation. Qualitatively, fractions M1 and M2 show impressive removal of erythrocytes, leukocytes and debris. Quantitatively, enrichment factors vary from 0.1 (depletion) to as high as 26 yielding an average enrichment of about 6, a most rewarding improvement.
A volume-activated cell sorter.
Authors: Menke E , Kordwig E , Stuhlmüller P , Kachel V , Ruhenstroth-Bauer G
Published: April 2011

A sorter that is activated by the resistance pulses of a Coulter orifice (volume detector) has been developed. The problem arising from the distortion of the volume signals produced by the electric droplet forming and charging voltages are avoided by special shielding and grounding of the volume detector. Experiments with beads and leukocytes are described. The maximal processing rate at this time is 2000 cells/sec, the purity of sorted fractions is better than 97%, and the viability of sorted cells is better than 80%.
The automation of cytochemical methods for automated cytophotometers.
Authors: Saunders A M
Published: April 2011

The development of an automated differential white blood cell counter is reviewed. After the red cells have been lysed, the white cells are counted by staining and passing through an electro-optical chamber in liquid suspension, surrounded by a laminar, or sheath, stream. Staining procedures were made specific for each type of leukocyte, and separate channels were used for counting each type. Staining intensity and characteristics of the various types of blood cells are discussed. They relate to enzyme levels and the effect on differentiation and identification of the cells. Since reasons for some of the design features are not obvious, discussion of the relevant problems is included. Several applications that go beyond routine differential counting are described.
Immunocytochemical localization of epidermal growth factor in mouse submandibular gland.
Authors: Gresik E , Barka T
Published: April 2011

The cellular and subcellular localization of epidermal growth factor in the submandibular glands of male and female adult mice was established by immunoperoxidase techniques. In light microscopic preparations epidermal growth factor was found exclusively in the granular convoluted tubules of the gland. The intensity of staining for epidermal growth factor varied from cell to cell, and some cells apparently were negative. The pattern of staining was similar in the glands of male and female mice; however, the granular convoluted tubules are androgen-responsive, and thus more extensive and composed of larger cells in males. In thin sections epidermal growth factor was most heavily concentrated in the secretion granules of the granular convoluted tubule cells. Within a given cell there was variation in intensity of staining of individual secretion granules, with some granules appearing minimally reactive or negative. The only other cell component with deposits of reaction product was the ribosomes.
Immune complex receptors on cell surfaces. III. Topography of macrophage receptors demonstrated by new scanning electron microscopic peroxidase marker.
Authors: McKeever P E , Spicer S S , Brissie N T , Garvin A J
Published: April 2011

Receptors for immune complexes have been localized on rabbit alveolar macrophages with scanning electron microscopy by exposing the cells first to a soluble immune complex composed of horseradish peroxidase and antibody to horseradish peroxidase, and then incubating with a benzidine-containing substrate that yields crystalline reaction product. Receptors were visualized by this means as sites of attachment of laminated slender crystals that were easily distinguished from macrophage surface structures. Receptors appeared most abundant on cytoplasmic veils and pseudopods and in the perinuclear region of macrophages minimally spread over the coverslip. Further macrophage spreading was associated with lighter receptor staining.
Evaluation of the utility of air-dried whole mounts for quantitative electron microprobe studies of platelet dense bodies.
Authors: Costa J L , Tanaka Y , Pettigrew K , Cushing R J
Published: April 2011

A variety of electronmicroscope techniques have been used to examine how the air-drying process may affect the dense bodies in whole mounts of platelets. (a) Selected-area-diffraction and electron microprobe studies suggest that the air-drying process can result in the formation of crystalline precipitates of sodium chloride on grid films and platelets. However, no crystals were detected in the calcium-and-phosphorus-containing matrix of dense bodies. (b) Tilting studies show that dense bodies in human platelets are spherical or ellipsoidal in shape. Dense bodies in rabbit platelets, in contrast, appear flattened in a horizontal plane. (c) Human-platelet dense bodies probed with a small (20 nm diameter) spot vary widely in their peak/background ratios for calcium and phosphorus-a finding that suggests that the two elements may not be evenly distributed throughout the dense-body matrix. Nevertheless, when dense bodies are probed with a larger (200 nm diameter) spot, they do not appear to differ appreciably among themselves in their calcium or phosphorus content. The data suggest that with human platelets, air drying may be a preparative procedure which permits comparison by microprobe techniques of dense-body matrix content in platelet populations.
On the lack of specificity of the cobalt-bicarbonate method for carbonic anhydrase.
Authors: Muther T F
Published: April 2011

Data are presented that support a nonenzymic mechanism for the staining obtained with the cobalt-bicarbonate method. The biochemically inactive apocarbonic anhydrase and Cu+2 apocarbonic anhydrase stain positively and this stain is inhibited by acetazolamide. The staining of the acetazolamide resistant carbonic anhydrase of male rat liver is inhibited by 10-6 M acetazolamide, at which concentration no biochemical inhibition is observed. There is no correlation between the biochemical and histochemical inhibitory potencies of a number of sulfonamides. The nonsulfonamide inhibitor, KCNO, does not inhibit staining. When incubations are performed in media exposed to atmospheres of increasing CO2 content, staining is not abolished until the atmospheric pCO2 approaches that generated by the medium itself. This finding renders the carbonic anhydrase catalyzed dehydration of HCO3- an improbable reaction for the staining. Studies with modified media show differences in staining patterns and in sensitivity to acetazolamide inhibition which question the specificity of the method for carbonic anhydrase.
A moving spot high resolution scanning microspectrometer using focused laser illumination: illustrations with acriflavin stained sephadex beads and human fibroblast nuclei.
Authors: Salmeen I , Rimai L , Levinson J W , McCormick J J
Published: April 2011

We describe a high resolution moving spot scanning microspectrometer, capable of absorption or fluorescence detection, using focused laser illumination which is moved over the sample by rotating the laser beam direction prior to focusing. This rotation is achieved by reflecting the beam from mirrors mounted on bending mode piezoelectric transducers which, when bent by an applied voltage, cause the mirrors to rotate. The images of optically thin samples are analyzed by considering the convolution of the focused spot intensity distribution with the absorbance of a uniformly stained spherical particle. This analysis is verified experimentally with data from acriflavin stained Sephadex beads. Data from acriflavin-Feulgen stained human fibroblasts indicate that the efficiency of this type of nuclear staining is about 2 to 3 dye molecules incorporated per 100 nucleotide pairs. Quantitative data on fading of acriflavin fluorescence in stained fibroblasts indicate that fading is negligible in the time required to record the microscope images.
Fixation variables in horseradish peroxidase neurohistochemistry. I. The effect of fixation time and perfusion procedures upon enzyme activity.
Authors: Rosene D L , Mesulam M M
Published: April 2011

In a series of neurohistochemical experiments the effect of aldehyde fixation upon the detection of horseradish peroxidase (HRP) was examined. These experiments demonstrated that: a) Increments in fixation of as little as 1 hr significantly decreased the number of labeled neurons; 12-hr fixation abolished HRP activity in many neuronal populations and significantly reduced the apparent size of the injection site. b) This negative fixation effect was greatest where the HRP concentration was low (e.g. in small, lightly labeled neurons) but was still evident in areas of high concentration (e.g. large, heavily labeled neurons). c) This effect was also most prominent when a less sensitive diaminobenzidine histochemical procedure was employed but was still apparent with a more sensitive benzidine dihydrochloride procedure. d) Immersion of the brain in fixative after perfusion produced a greater attenuation of HRP activity in more superficial areas. e) Immersion of the brain in buffer to terminate fixation produced a prolonged and unpredictable gradient of fixation. f) Excess, unbound fixative inhibited the histochemical reaction per se and had to be removed from the tissue but prolonged washing did not resurrect enzyme activity which was lost by fixation. To obviate these problems and optimize HRP enzyme activity a new perfusion-fixation procedure was developed. It entails 30 min fixation by perfusion which is terminated by a subsequent 30 min perfusion with cold sucrose-fuller to wash out unbound fixative. This allows the tissue to be processed immediately, produces a uniform and morphologically adequate fixation, and minimizes the negative effects of fixation on HRP enzyme activity.
Pyridoxal phosphatase: cytochemical localization in GERL and other organelles of rat neurons.
Authors: Spater H W , Novikoff A B , Spater S H , Quintana N
Published: April 2011

A phosphatase, hydrolyzing pyridoxal-5-phosphate (P5P), a physiologically active component of the vitamin B6 complex and an essential co-enzyme in the synthesis of neurotransmitters, has been localized cytochemically in the perikarya of neurons in the peripheral, autonomic and central nervous systems of the rat. Neurons in dorsal root ganglia, sympathetic ganglia and ventral horn of spinal cord were studied by light and electron microscopy, while Purkinje cells, neurons in the dentate nucleus of the cerebellum, thalamus, and hypothalamus were studied by light microscopy only. Optimal conditions for demonstrating this activity in aldehyde-fixed tissue were determined with dorsal root ganglia. At the optimal pH of 5.0, neurons in these ganglia and in all other neurons studied show pyridoxal-5-phosphatase (P5Pase) activity in GERL. Small neurons in dorsal root ganglia also display enzyme activity in the endoplasmic reticulum (ER); activities in GERL and ER are also appreciably high at neutral pH. Small and large neurons in these ganglia, and neurons of sympathetic ganglia, show variable P5Pase activity in the Golgi apparatus. These localizations differ from the usual sites of both acid phosphatase and alkaline phosphatase activities. The P5Pase activity, demonstrated cytochemically, is a new acid hydrolase activity in GERL.
Studies on acid lipase, and E 600-resistant acid esterase activities in human tissue homogenates.
Authors: Young C W , Mladek J , Smith S W , Bittar E S
Published: April 2011

Detailed comparison of acid lipase and acid esterase activities of human spleen, liver and kidney homogenates has been carried out by means of the following substrates: 14C-tripalmitin, alpha-naphthyl acetate, alpha-naphthyl butyrate, alpha-naphthyl laurate, p-nitro-phenyl acetate, butyrate and laurate. In addition, homogenates of the three tissues were subjected to isoelectric focusing in polyacrylamide gels and histochemical staining with the above mentioned naphthyl substrates in the presence and absence of the organophosphate esterase inhibitor diethyl-p-nitrophenyl phosphate (E 600). These studies provide extensive support for the proposal that E 600-resistant acid naphthyl butyryl and lauryl esterase activities in human tissues derive largely from the enzyme acid lipase. The studies suggest that the most specific chromogenic substrate for this enzyme at a biochemical and histochemical level is alpha-napthyl laurate in the presence of E600 (3 X 10(-6) M).
Device for quantitative cytochemistry: a computerized scanning microdensitometer 'grain counter'.
Authors: Markovic N S , Lipkin L E , Markovic O S , Wade M J
Published: April 2011

A computerized scanning microdensitometer and autoradiographic grain counter was able to provide quantitative data on the cytochemical final reaction product formed within a single cell and also quantitate the kinetics of its formation. Optical density and area measurements were performed on hundreds of leukocytes from slides previously stained to demonstrate any one of a variety of reactions. These included cellular glycogen, lipids, peroxidase, esterases, alkaline phosphatase, and acid phosphatase. In addition to these slide studies, chamber studies with an adapted Dvorak-Stotler Chamber allowed the measurement of enzyme kinetics within single cells.
Ultrastructural localization of alkaline phosphatase in rat eosinophil leucocytes.
Authors: Williams D M , Linder J E , Hill M W , Gillett R
Published: April 2011

The ultrastructural localization of alkaline phosphatase in eosinophil leucocytes, obtained from experimentally-induced peritoneal exudates in rats, has been studied using an osmiophilic technique with 2-naphthylthiolphosphoryl dichloride as substrate, fast Blue BBN as diazonium salt and postosmication with 1% aqueous osmium tetroxide. With this method identical incubation procedures could be used for both light and electron microscope examination. Eosinophils were the only cells which contained alkaline phosphatase. The enzyme was predominantly associated with the outer surface of the plasma membrane, being present in much lower concentrations in cytoplasmic cisternae. Eosinophil granules only rarely showed reaction product. The plasma membrane location of alkaline phosphatase in eosinophil leucocytes is identical to that recently demonstrated in the human neutrophil.
Immunofluorescent staining of histaminase (diamine oxidase) in human placenta.
Authors: Lin C W , Chapman C M , DeLellis R A , Kirley S
Published: April 2011

An immunofluorescent procedure for the localization of histaminase in human tissue sections has been developed by using a specific antiserum against human placental histaminase. For localization of this enzyme in placental sections, fixation in equal volumes mixture of absolute ethanol and acetone provided the optimum visualization of this enzyme in both frozen sections and paraffin-embedded sections. The immunofluorescent staining of this enzyme in placenta is found to be localized in areas within the maternal decidua, both within the cytoplasm of the decidual cells and in tissue space between the cells. The chorionic villi are completely void of the immunofluorescent stain. Variations in patterns of histaminase localization have been found between term and premature placentas, with the former showing a predominantly intercellular localization and the latter a predominantly intracellular localization. The intercellular localization of this enzyme in the decidua may represent a nonspecific diffusion of the enzyme associated with delivery of the placenta or may reflex a specific functional role of the enzyme in the intercellular space during pregnancy.
Histochemistry of rat intrafusal muscle fibers and their motor innervation.
Authors: Kucera J , Dorovini-Zis K , Engel W K
Published: April 2011

Muscle spindles were followed in serial transverse sections of freshly frozen rat soleus muscles. Adenosine triphosphatase (ATPase) histochemical staining reaction was used to identify nuclear bag1, nuclear bag2 and nuclear chain intrafusal muscle fibers. Regional differences in ATPase staining occurred along bag1 and bag2 fibers but not along chain fibers. Bag1 fibers displayed ultrastructural heterogenity when their intra- and extracapsular regions were compared. Simple "diffuse" and more elaborate "plate" motor nerve terminals were demonstrated histochemically along the poles of bag1 and bag2 fibers by staining for cholinesterase. One motor terminal of the "plate" appearance was present on a chain fiber pole. There was no consistent spatial correlation between the intensity of regional ATPase staining along the nuclear bag fibers and the location, number and type of motor endings. Other factors, such as intrafusal fiber sensory innervation and regional differences in active and passive functional recruitment of nuclear bag fibers during muscle activity, may contribute to the ATPase staining variability along the intrafusal fibers.
Detection of tubulin and actin in various cell lines by an immunoperoxidase technique.
Authors: Karsenti E , Guilbert B , Bornens M , Avrameas S , Whalen R , Pantaloni D
Published: April 2011

This paper reports on the preparation of immunsera against tubulin and actin, and the purification of anti-tubulin and anti-actin antibodies on immunoadsorbent columns. These purified antibodies were used in an indirect immunoperoxidase assay to visualize microtubules and microfilaments in various cell lines. The specificity of antibodies and the methods of cell fixation required are discussed, as well as some aspects of microtubule and microfilament organization, as visualized by this technique.
Computer analysis of cervical cells. Automatic feature extraction and classification.
Authors: Holmquist J , Bengtsson E , Eriksson O , Nordin B , Stenkvist B
Published: April 2011

A prescreening instrument for cervical smears using computerized image processing and pattern recognition techniques requires that single cells in the specimen can be automatically isolated and analyzed. This paper describes a dual wavelength method for automatic isolation of the cytoplasm and nuclei of cells. Density-oriented, shape-oriented and texture-oriented parameters were calculated and evaluated for more than 600 cells. It is shown that the computer can be taught to distinguish between normal and atypical cells with an accuracy of ca. 97%, while human classification reproducibility is ca. 95%. In addition, an attempt to assign a measure of atypia to individual cells is described.
Simultaneous localization of an hepatic binding protein specific for galactose and of galactose-containing receptors on rat hepatocytes.
Authors: Horisberger M , VonLanthen M
Published: April 2011

The hepatic binding protein, specific for galactose-terminated glycoproteins (asialoglycoproteins) and the receptors for the Ricinus communis lectin, specific for galactose residues (RCA1), were simultaneously localized on isolated rat hepatocytes by the gold method. The marker for the binding protein was prepared from gold granules (5 nm in diam.) labeled with ceruloplasmin and desialylated. The marker specific for galactose-containing receptors consisted of granules (17 nm in diameter) labeled with RCA1. It was established that both markers did not interact. Hepatocytes (fresh or briefly fixed with glutaraldehyde) were successively incubated with the asialoceruloplasmin and the RCA1 marker. Examination of thin sections by electron microscopy indicated that the binding protein and the RCA1 receptors were often in the proximity of each other on the plasmamembrane. Using the same technique, wheat germ agglutinin (WGA) receptors were generally found on area of the plasmamembrane poorly marked by the RCA1 gold marker. The binding of asialoceruloplasmin gold markers was studied as a function of the size of the granules. It became insignificant when the size was above 17 nm. Previous results have shown that the binding of RCA1 is low when the marker reaches 50 nm in size while WGA markers up to 75 nm are well bound by hepatocytes. It is therefore hypothesized that the binding protein and RCA1 receptors are located between glycoprotein brushes of increasing spacing while part or all of the WGA receptors are located at the periphery of the brushes.
Endogenous thyrotropin-releasing hormone in the anterior pituitary: sites of activity as identified by immunocytochemical staining.
Authors: Childs G V , Cole D E , Kubek M , Tobin R B , Wilber J F
Published: April 2011

Thyrotropin-releasing hormone (TRH) immunoreactivity was localized in the rat anterior pituitary with rabbit anti-TRH sera and the unlabeled antibody peroxidase-antiperoxidase complex (PAP) technique. Stain was present in secretory granules of cells possessing morphological characteristics of thyrotropes, gonadotropes and lactotropes. Antibody absorption studies with anti-TRH sera absorbed with TRH, 3 diastereoisomeric analogues of TRH, gonadotropin-releasing hormone (GnRH), bovine serum albumin, thyrotropin, prolactin, adrenocorticotropin, luteinizing hormone, follicle stimulating hormone were performed to determine the specificity of the staining reaction. Only absorption with TRH resulted in a significant reduction in staining intensity. In vitro experiments were then begun with hemipituitaries to ascertain if intrapituitary TRH might originate by sequestration of exogenous, plasma membrane bound TRH or by de novo synthesis. The results suggest that anterior pituitary TRH is of endogenous origin.
Masking of protein antigen by modification of amino groups with carbobenzoxychloride (benzyl chloroformate) and demasking by treatment with nonspecific protease.
Authors: Takamiya H , Bodemer W , Vogt A
Published: April 2011

Cryostat sections of various substrates were treated with carbobenzoxychloride in acetone to modify antigens. By applying specific fluorescent antibodies, it could be shown that the antigenic determinants of rabbit gamma-globulin and bovine insulin were totally masked. The antigenicity of ACTH was markedly reduced, whereas the polysaccharide antigens of Salmonella typhimurium were only partially masked. After masking, antigenicity could be restored by treatment with nonspecific protease. The reversible protection of amino groups by carbobenzoxychloride may be a way to preserve protein antigens during embedding in plastics, as such materials also bind to amino groups, blocking the antigenicity of proteins.
A kinetic study of the reaction between glutaraldehyde and horseradish peroxidase.
Authors: Molin S O , Nygren H , Dolonius L , Hansson H A
Published: April 2011

Horseradish peroxidase was reacted with glutaraldehyde under various reaction conditions. The reaction product was, in a second step, bound covalently to aminohexyl groups attached to Sepharose particles. The influence of pH, time and the concentration ratio of enzyme:glutaraldehyde on the reaction was evaluated. A first step reaction with 100-fold molar excess of glutaraldehyde to horseradish peroxidase at pH 9.5 for 2 hr at room temperature results in a high yield of conjugated enzyme with well preserved enzymatic activity.
Ultrastructural localization of intracellular antigens by the use of protein A-gold complex.
Authors: Roth J , Bendayan M , Orci L
Published: April 2011

An immunocytochemical technique for the demonstration of intracellular antigens (secretory proteins) on thin sections is reported. Staphylococcal protein A which reacts with the Fc fragment of IgG molecules was labeled with colloidal gold as a marker. The antigenic sites were visualized on aldehyde-fixed and Epon-embedded tissue in a two step procedure. The specific antisera were applied to thin sections for binding to the antigens and then visualized by the protein A-gold complex. By using this technique different secretory proteins of the exocrine and endocrine pancreas were localized. The protein A-gold technique is proposed as a general method for visualization of antigenic sites on thin sections.
Inhibition of epithelial cell death in the secondary palate in vitro by alteration of lysosome function.
Authors: Greene R M , Pratt R M
Published: April 2011

The secondary palate in vivo and in vitro exhibits selective cell death at its medialedge epithelium (MEE) at a precise developmental age. This epithelial degeneration is mediated, in part, by MEE lysosomes. Previous studies in vitro (27) showed that the glutamine analogue, diazo-oxo-norleucine (DON), prevented MEE cell death by inhibiting glucosamine synthesis and thereby the glycosylation of proteins without affecting either the synthesis or activity of palatal lysosomal enzymes. In the present study, histochemical examination of MEE from DON treated day-15 rat palates demonstrated that acid phosphatase activity was restricted to Golgi saccules and associated vesicles as well as to lysosomes. Control MEE had reaction product in these structures and distributed diffusely throughout the cytoplasm of degenerating cells. DON treatment therefore appears to alter the intracellular distribution of lysosomal enzymes. Since DON treatment appears to have prevented MEE cell death by inhibiting glycosylation of proteins, glycosylation of lysosomal membranes or lysosomal enzymes may be essential for its role in programmed cell death.
Atrial specific granules of the rat heart: light microscopic staining and histochemical reactions.
Authors: de Bold A J , Raymond J J , Bencosme S A
Published: April 2011

An investigation was carried out to determine general staining and histochemical properties of rat atrial specific granules. It was found that these granules may be demonstrated using aldehyde fuchsin after pretreatments which involve oxidation or thiosulfation. This new way of demonstrating atrial granules is compared to other staining methods in terms of sensitivity and selectivity as well as to the nature of reactive groups that may be involved in the staining reactions. No lipid or carbohydrate were detected histochemically. Overall assessment of reactions suggests that atrial granules are a site of storage for a protein or polypeptide. Some of the tests indicate that these may contain tryptophan and sulfur-containing amino acids.
In situ embedding method of cells grown in beem capsules for immunoelectron microscopic studies of oncornaviruses.
Authors: Ohtsuki Y , Dmochowski L , Seman G , Bowen J M
Published: April 2011

A technique of in situ embedding of cells grown in BEEM capsules has been devised for immunoelectron microscopic studies of oncornaviruses. As compared to other immunoelectron microscopic procedures, this technique is less time and reagent-consuming. The quality and specificity of this method were tested on well-characterized mouse mammary tumor virus (type B virus) and murine sarcoma virus (type C virus particles). This method gave good results in labeling of the virus particles with ferritin or peroxidase in the cells of mouse tissue cultures. In an application of this method, peroxidase labeling of type B virus particles was obtained in frozen sections of normal prostatic tissues of C3H/Dm and A/Dm strain mice treated with rabbit antiserum to mouse mammary tumor virus from A/Dm strain mouse milk. These results indicate that this method is useful and reliable for immunoelectron microscopy studies of oncornaviruses in tissue culture cells and also in frozen sections of tissues.
Mast cell granule formation in the beige mouse.
Authors: Chi E Y , Ignácio E , Lagunoff D
Published: April 2011

The formation of mast cell granules was studied in the beige mouse utilizing histochemistry and electron microscopy. The time and sequence of appearance of heparin, histamine and the chymotrypsin-like protease were normal. By electron microscopy, the initial formation of progranules and subsequent aggregation was normal, but the granules from early stages were abnormally large. Reorganization of intermediate granule forms to homogeneous mature granules was delayed. Late fusions of intermediate and/or mature granules were not observed. Our findings indicate that the defect lies in the excessive initial fusion of progranules rather than in continued formation of new progranules or in fusion of mature granules with one another.
Studies on the secretory process in exocrine pancreas cells. II. C57 black and beige mice.
Authors: Novikoff A B , Quintana N , Mori M
Published: April 2011

Published electron microscopic and cytochemical studies (thiamine pyrophosphatase and acid phosphatase) on exocrine pancreas cells of guinea pig, hamster, rat and rabbit have demonstrated that the nascent secretory granules, or condensing vacuoles, are part of GERL. The studies reported here show this to be true of the mouse pancreatic exocrine cells as well, thus permitting comparison of this cell type in the C57 black mouse and its "beige" mutant. This is of considerable interest because GERL is very much enlarged in these cells of the beige mouse. Most of GERL consists of wide dilated portions filled with electron-opaque materials that appear to be packaged into huge residual body-type lysosomes ("anomalous granules"). Acid phosphatase activity is demonstrable not only in these portions of GERL, but also in the condensing vacuoles as in pancreatic acinar cells in the black mouse where these dilated lysosome-producing regions are not present.
Coloidal gold, ferritin and peroxidase as markers for electron microscopic double labeling lectin techniques.
Authors: Roth J , Binder M
Published: April 2011

Three markers, colloidal gold, ferritin and peroxidase, were checked for usefulness in double labeling of lectin-binding sites. The amount of various lectins for the stabilization of good sols of a different particle size was evaluated. Several lectin-gold complexes were prepared for electron microscopic labeling purposes, and the optimal amount of various lectins needed for stabilization of gold solutions of a different particle size was determined. The following combinations were investigated for their usefulness in labeling two different lectin-binding sites: lectin-gold and lectin-gold (different particle size), lectin-gold and lectin-ferritin, as well as lectin-ferritin and lectin-peroxidase. Of these combinations the latter did not give satisfactory results for double labeling. In all single and double labeling techniques with the above mentioned markers the quantitative evaluation of the number of lectin-binding sites is not feasible, but these techniques will be of considerable value for the investigation of the dynamics of different lectin-binding sites on the cell surface.
Acid D-ephedrine-O-phosphatase activity in rate ventral prostate lysosomes.
Authors: Schofield B H , McDonald D F , Geffert M A
Published: April 2011

Hydrolysis of D-ephedrine-O-phosphate (DEP) by rat ventral prostrate was investigated by electron microscopic hystochemistry. In contrast to previous reports that DEP is exclusitely hydrolyzed by a secreted nonlysosomal acid phosphatase, enzyme reaction product was present in supranuclear and basal lysosomes.
Fluorescent antibodies and lectins stain intracellular structures in fixed cells treated with nonionic detergent.
Authors: Laurila P , Virtanen I , Wartiovaara J , Stenman S
Published: April 2011

Nonionic detergent (NP40) treatment of paraformaldehyde-fixed normal and SV40-transformed human fibroblasts resulted in intracellular penetration of two chosen fluorescent antibodies and Concanavalin A (Con A). After the detergent treatment nuclear SV40 T antigen, cytoplasmic fibronectin glycoprotein and Con A binding sites could be visualized in fluorescence microscopy. The lowest NP40 concentration which made fixed cells permeable was 0.05%. The morphology of cells was preserved better by this new method than by conventional fixation methods, such as acetone treatment. In scanning electron microscopy the surface of the fixed NP40-treated cells had only small rugosities and fine pores. The subsurface cytoskeleton especially was well preserved and had a more distinct fine structure. The improved morphology made it possible to detect a similar distribution of fibronectin and Con A binding sites in the perinuclear endoplasmic reticulum regions.
Histochemical differentiation of complex carbohydrates with variants of the concanavalin A-horseradish peroxidase method.
Authors: Katsuyama T , Spicer S S
Published: April 2011

Various treatments carried out prior to the concanavalin A-horseradish perioxidase (HRP) method have been found to affect the staining and have permitted differentiation of three main classes of complex carbohydrates in the rat alimentary tract. Class I mucosubstances lose and class II and III paradoxically gain concanavalin A-horseradish peroxidase reactivity after periodate oxidation. Class II mucosubstances lose whereas class III retain or increase their reactivity with a reduction step interposed between oxidation and concanavalin A-horseradish peroxidase staining. Mucous neck cells, pyloric glands, Brunner's glands and mast cells exhibit strong class III staining, whereas other sites such as intestinal goblet and salivary gland acini differ widely in their type of staining. Liver glycogen stains like mucosubstances in an unstable subgroup of class III. The paradoxical increase in concanavalin A binding during oxidation correlates with the appearance of Schiff reactivity implicating oxidation of vicinal hydroxyls as the basis for the effect. The periodate-induced staining is therefore, thought to result from an oxidative disruption of linkages between vicinal hydroxyls of neighboring sugars and hydroxyls of mannose required for concanavalin A binding. Staining with the described concanavalin A-horseradish peroxidase variants appears to afford information concerning cytochemical distribution of mannose-rich glycoproteins as well as differences among these substances in the relation of mannose to neighboring sugars.