bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunocytochemical methods in the study of lymphoma and related conditions.
Authors: Taylor C R
Published: April 2011

The diagnosis and classification of malignant lymphomas is based upon traditional histological criteria evolved over the years by successive generations of pathologists. Immunoperoxidase methods applied to formalin paraffin sections have permitted a direct correlation of these established morphologic criteria with newer immunological concepts of the form and function of the B lymphocyte and its derivatives. Study of material from 500 cases of Hodgkin's disease, non-Hodgkin lymphomas, and myeloma has revealed that many of these conditions may find a common origin in the lymphocyte, and that some malignant cells, previously identified as malignant histiocytes or reticulum cells are rather related to or derived from the transformed lymphocyte or immunoblast. The corresponding tumors are thus more logically designated immunoblastic sarcoma. The study also reveals a claser developmental relationship between multiple myeloma and immunoblastic sarcoma than previously suspected, and suggests that all of the B cell lymphomas occur as part of a continuous spectrum of disease, rather than as separate entities as implied by current histological classifications. The study of immunoglobulin in formalin paraffin sections is illustrative of the great potential of this method in the diagnosis and study of neoplasia in general.
Immunocytochemical localization of oncodevelopmental proteins in human germ cell and hepatic tumors.
Authors: Palmer P E , Wolfe H J
Published: April 2011

In a combined tissue and serum study alpha-1-antitrypsin (AAT) and alpha-fetoprotein are demonstrated in parallel within tumor tissue inclusions in both endodermal sinus (yolk sac) tumors and malignant hepatomas, and AAT is demonstrated as a marker in both neoplastic and preneoplastic liver lesions occurring in oral contraceptive users, all in association with normal serum AAT phenotype. The tumor inclusions in the first two instances differ immunocytochemically from AAT liver cell globules found in inherited AAT deficiency, which are unreactive for alpha-fetoprotein. It is concluded that unlike the molecular basis of storage associated with AAT phenotypic variation, the tumor inclusions reflect a separate, nongenetic mechanism of AAT storage, which may be epigenetic in nature. AAT and alpha-fetoprotein both are synthesized normally in yolk sac and fetal liver, a parallelism which disappears soon after birth. The reexpression of both proteins in two distinct tumor types arising from endodermal origins (yolk sac and liver), suggests that these markers may represent reemerging fetal gene products, a phenomenon previously proposed only for alpha-fetoprotein, a prototypic "oncofetal antigen."
High resolution dual laser flow cytometry.
Authors: Dean P N , Pinkel D
Published: April 2011

Flow cytometers based on optical sensing utilize external light sources and fluorescent dyes to measure one or more specific components or properties of individual cells or subcellular particles in liquid suspension. To provide for independent excitation of two dyes used in double staining experiments we have constructed a high resolution flow cytometer that uses two laser beams to provide two wavelengths of excitation. These beams are separated spatially so that cells flow through them sequentially, with a time separation of about 20 musec. Since the dyes are excited sequentially their emission occurs at different times and their emission spectra may overlap without causing any difficulty in analysis. We have developed new light collection optics that permit up to four measurements to be made on each cell. This approach greatly increases the number of dye combinations that can be used in flow cytometry, thus removing a significant limitation of single illumination instruments.
Use of horseradish peroxidase to block nonspecific enzyme uptake in immunoperoxidase microscopy.
Authors: Minard B J , Cawley L P
Published: April 2011

Intrinsic tissue peroxidase activity can be more or less successfully destroyed by methanol-H2O2 treatment. It has been found, however, in our laboratory that horseradish peroxidase (HRP) coupled to antibody will bind to some tissue components on a nonspecific basis and remain to take part in the histochemical stain. This contributes considerably to the background. This difficulty can be largely overcome if the tissues are pretreated with a solution of horseradish peroxidase which binds with nonspecific tissue sites. The adsorbed enzyme, along with the intrinsic peroxidase, can then be successfully inactivated by methanol-H2O2 treatment. By this method of blocking, there is considerable reduction in background staining.
On the quantitation of SV40 virus by plaque assay and immunoperoxidase technique.
Authors: D'Alisa R M , Gershey E L
Published: April 2011

Procedures are described for the quantitation of SV40 virus infectivity by plaque formation within 7 days and T antigen assay by the sensitive and economical indirect immunoperoxidase technique.
A photographic technique to increase image contrast in photomicrographs.
Authors: Grigonis G J
Published: April 2011

The photographic technique described in this report enables investigators to emphasize image details in low contrast photographs, resulting from under exposure, poor development or low contrast subject matter. The technique is easily carried out in nearly any darkroom facility and is less costly and faster than the more sophisticated methods available today.
Differences in flow cytometry and 3H-thymidine analyses of perturbed human lymphocytes.
Authors: Pollack A , Bagwell C B , Hudson J L , Irvin G L
Published: April 2011

A calf thymocyte crude aqueous extract was tested for DNA synthesis inhibitory activity using phytohemagglutinin-stimulated human peripheral blood lymphocytes. Inhibition of DNA synthesis was assayed using tritiated thymidine and flow cytometry. Although the calf thymocyte crude extract inhibited tritiated thymidine incorporation by over 50%, only very slight changes in the flow cytometric analysis were observed. When dibutyryl-cyclic adenosine monophosphate was used as an inhibitor, a correlation in terms of the inhibition of tritiated thymidine to the inhibition by flow cytometry was observed.
Flow microfluorometric identification of liver cells with elevated gamma-glutamyltranspeptidase activity after carcinogen exposure.
Authors: Vanderlaan M , Cutter C , Dolbeare F
Published: April 2011

We have developed a fluorometric cytochemical assay for gamma-glutamyltranspeptidase (gamma-GT) using the substrate gamma-glutamyl-4-methoxy-2-naphthylamide in which the released methoxynaphthylamine was coupled with 5-nitrosalicylaldehyde to form a yellow fluorescent crystalline product within the cells. Single cell suspensions were obtained by collagenase perfusion of livers from rats that had either received a two-thirds partial hepatectomy followed 24 hr later by a single injection of diethylnitrosamine (DEN) or received a partial hepatectomy alone. Cultured HTC cells were used as a source of gamma-GT+ cells. Fluorescence (gamma-GT activity) was low in most of the cells from both DEN-exposed and control rats, but high in HTC cells. The livers of both DEN-exposed and control rats had a subpopulation of cells that were gamma-GT+; this population could be quantitated and sorted by flow cytometry. Five weeks post injection the number of GT+ cells from the rats exposed to DEN was more than 20 times that from the control rats. Increased gamma-GT activity may be a useful cytochemical marker for preneoplastic liver cells.
Ultrafast laser scanner microscope.
Authors: Shack R , Baker R , Buchroeder R , Hillman D , Shoemaker R , Bartels P H
Published: April 2011

Advances in monolayer deposition of cervical cells have removed one of the last serious obstacles to the design of high-resolution automated diagnostic assessment systems. In this article, we describe the design considerations for a system that is capable of acquiring, within 60 sec, a 0.5 micron digitized image of a 4 cm2 area on a standard glass slide. The most feasible approach is found to be a system using a rotating polygon to sweep the focused spot from a laser across a 2-mm scan line while the slide is uniformly translated perpendicular to the scan direction the use of laser sources (a helium-neon laser at 632 nm and a krypton ion laser at 568 and/or 476 nm) as compared to the incoherent light sources used in conventional microscope systems alleviates many of the optical design problems and provides the proper wavelengths needed for recognition of Papanicolaou stained cells. We also find that focus control of the scanning spot should be achievable using a technique involving a holographic grating. Other relevant considerations such as sample heating problems, multiphoton absorption by the sample, detector signal-to-noise ratios, laser amplitude noise control, and the digitization and buffering of the data stream are also discussed.
Light scattering with stream-in-air flow systems.
Authors: Salzman G C , Wilder M E , Jett J H
Published: April 2011

Both forward angle and 90 degrees light-scattering measurements have been used for cell sizing with stream-in-air flow systems with very little theoretical base for the measurements. Mie theory calculations are compared with measurements on plastic microspheres. Detector response for homogeneous spheres is shown to be sensitive to refractive index.
A multidimensional slit-scan flow system.
Authors: Cambier J L , Kay D B , Wheeless L L
Published: April 2011

A new slit-scan type flow system is described which provides three (X, Y, and Z) orthogonal one-dimensional projections of cell fluorescence. A photomultiplier tube and two semiconductor array detectors are used to obtain the three slit-scan contours from cells traversing a single fluorescence excitation beam. A high speed, dedicated preprocessor analyzes the three contours in parallel, extracting certain features useful for rejecting cells from which an accurate measurement of nuclear fluorescence cannot be obtain. Contour data is buffered and transferred to a PDP-11/40 computer where nuclear fluorescence is measured and cells are classified. It is anticipated that this new instrument will provide a significant reduction in false alarm rate when applied to prescreening of gynecologic cytology specimens.
Fast imaging in flow: a means of combining flow-cytometry and image analysis.
Authors: Kachel V , Benker G , Lichtnau K , Valet G , Glossner E
Published: April 2011

The morphological identification of cells by flow cytometry is difficult. Usually cell sorting and microscopical analysis have to be used in addition. Morphological analysis is simplified by taking cell pictures from a range of particular interest immediately during flow cytometric analysis. Instruments using the video scanning technique for fluorescence imaging are slow and expensive (8, 10). Morphological information can also be obtained by transmission imaging of cells in flow, which requires shorter exposure times. Therefore a cell volume activated flow imaging device has been developed which operates at flow speeds up to 5 m/sec and which depicts transmission images of selected cells on a 16-mm film by a nsec flashlamp illumination. An electronic unit detects the particles in the optically accessible orifice, performs the pulse height analysis, triggers the flashlamp if particles are in the preselcted range of interest and feeds the film. The instrument is capable of delivering up to 150 pictures per second and works either as a flow microscope in which the cells in the preselected volume range are directly observed, or as a picture system in which the cell pictures are stored on the 16-mm film for documentation or for image analysis.
Flowcytometric evaluation of the DNA distribution in isolated pancreatic islets from normal and diabetic mice.
Authors: Larsen J K , Nielsen O
Published: April 2011

Pancreatic islets from C57BL/KsJ-db/db, and +/+ mice were isolated by collagenase. After isolation the islets were transferred to a hypotonic ethidiumbromide solution with 0.3% of the detergent Nonidet P40. After vortexing, the samples were analyzed in a BioPhysics Cytofluorograf 4802A. The DNA histograms were divided into 2c, 2-4c, 4c and 8c fractions under the assumption of a constant rate of DNA synthesis during the 2-4c phase. In comparison with normal mice, we found that diabetic mice had a lower fraction of 2c nuclei and a higher fraction of 2-4c, 4c, and 8c nuclei. These results obtained by flow cytometry are in agreement with results obtained by 3HTdR incorporation and by cytophotometry on histological sections.
Immunofluorescence of histone H1 in stimulated lymphocytes measured by flow cytophotometry.
Authors: Betz J W , Herman C J
Published: April 2011

The modification of histones or their redistribution during the transition from actively transcribing chromatin to the heterochromatic chromosomes seems to play a major in regulation of gene expression. The purpose of this study was to monitor the change in immunofluorescence of histone HI during phytohemagglutinin stimulation in peripheral lymphocytes. The histone antigens were prepared from pig thymus, proven to be pure by gel electrophoresis and repeatedly injected as RNA-complexes into rabbits. The antihistone HI antiserum titer was 1:4000, and there was no cross-reactivity with other histone fractions as shown by microcomplement fixation tests. Affinity chromatography purified antibody after being labeled with fluorescein isothiocyanate was able to differentially stain HeLa cells as controls and those, where histone HI had been extracted by perchloric acid treatment. The measurements were done on a Los Alamos Scientific Laboratories-flow cytophotometer cell sorter. Staining peripheral lymphocytes resulted in a bimodal distribution. The increase in number of cells with high fluorescence intensity had its maximum about 20 hr before the maximum proliferative activity of the lymphocytes as measured by number of cells in S phase with the DNA-stain mithramycin.
Efficient interaction for automated chromosome analysis using asynchronous parallel processes.
Authors: Piper J , Mason D , Rutovitz D , Ruttledge H , Smith L
Published: April 2011

It is likely that any practical automated chromosome analysis system will be interactive. To prevent long pauses in the stream of operator interactions, it is necessary, if using standard computer hardware, to configure for asynchronous and parallel operation. A system is presented which uses several computer processors, which can support one or more operators, and which divides processing into interactive and noninteractive sections, smoothes the rate of presentation of interactions, and keeps both the operator and the computer fully employed.
Isolated metaphase chromosomes. II. Proteins of Chinese hamster chromosomes.
Authors: Wray W , Wray V P
Published: April 2011

The proteins on metaphase chromosomes theoretically may be distributed ubiquitously throughout the karyotype, may be present uniquely on individual chromosomes or classes of chromosomes, or may exist in any combination of the above. Separation of chromosomes according to size using sucrose velocity gradients in high capacity zonal centrifuge rotors allows sufficient fractionation of the genome to indicate the distribution of proteins within the karyotype. Flow cytometric analysis and direct microscopic analysis were used to evaluate qualitatively the types of chromosomes present in the fractions obtained. This report is the first quantitative evidence that some of the chromosomal proteins are not distributed ubiquitously on all of the chromosomes of the karyotype.
An interactive computer system for the analysis of cell lineages.
Authors: Bell E , Levinstone D , Sher S , Marek L , Merrill C , Young I , Eden M
Published: April 2011

We have developed an interactive computer system for analysing cell lineage data. It can be utilized in studies of cell motility, cell division, cell differentiation, and cell aging. It has enabled us to document the heterogeneity of human foreskin fibroblasts in culture and to propose that loss of proliferative potential may mean that cells enter a state of differentiation which makes them unable to respond to mitotic stimulation. Our method, which enables us to apply immunological and cytochemical probes after recording the history of a cell lineage, should allow us to define precisely features which uniquely distinguish cycling from noncycling cells on an individual cell basis.
Cell sorter analysis of carcinogen metabolites in human tissues.
Authors: Tyrer H W , Adams L A , Tiffany S M , O'Connell J P , Cantrell E T
Published: April 2011

A fluorescence -activated cell sorter (FACS-II) was used to examine biochemical parameters in a heterogeneous population of cultured human lymphocytes. Incubation of cells in the presence of benz(a)anthracene (BA) during culture was employed to induce the enzyme system which metabolizes carcinogens. Carcinogen metabolism was assayed directly by measuring the phenolic metabolites of cells exposed to benzo(a)pyrene (BP). Metabolism of benzo(a)pyrene was measured in single cells and was determined to be greater in the larger cells than in the smaller cells of the cultures. For a given size of cells, the enzyme activity was greater in those exposed to benz(a)anthracene during culture. In some studies, viable cells were first sorted by size and subpopulations assayed for the o-deethylation of the compound, ethoxyresorufin, which measures more specifically the activity of cytochrome P-448. Larger cells had higher levels of enzyme activity than smaller cells in agreement with the direct determinations above. It is possible to measure carcinogen metabolism in other tissues by using the techniques described here.
An image analysis system for cervical cytology automation using nuclear DNA content.
Authors: Tucker J H
Published: April 2011

An experimental computer/image analysis system has been used to investigate cytology automation techniques based on nuclear DNA measurement and morphological artefact rejector tests. The system automatically measures and normalizes the integrated optical density of cell nuclei in specially prepared cervical cytology specimens, and selects any objects with abnormally high values for further analysis. These are then analyzed by morphological and densitometric tests designed to eliminate false positive signals caused by non-nuclear artefacts. The coordinates of the remaining abnormal nuclei are recorded so that they can subsequently be relocated and examined by a cytotechnician. Preliminary results are given showing the measurement accuracy of the system and the performance of the artefact rejection tests.
The preparation of cervical scrape material for automated cytology using gallocyanin chrome-alum stain.
Authors: Eason P J , Tucker J H
Published: April 2011

A method is described for preparing cervical scrape specimens for automated analysis on the Cerviscan prescreening system. In order to reduce the cellular clumping found in cervical scrape material, cells are collected in suspension, syringed to disaggregate the cell clumps, and then pipetted onto a glass to give a monolayer of cells. The cells are then stained with gallocyanin chrome-alum to give the required quantitation of nucleic acid content, using a rapid staining procedure. Experimental results are given which show that specimens prepared by this method are more suitable for automated analysis than the conventional Papanicolaou stained preparation.
Different sensitivity of chromatin to acid denaturation in quiescent and cycling cells as revealed by flow cytometry.
Authors: Darzynkiewicz Z , Traganos F , Andreeff M , Sharpless T , Melamed M R
Published: April 2011

The properties of DNA in situ as reflected by its staining with acridine orange are different in quiescent nonstimulated lymphocytes as compared with interphase lymphocytes that have entered the cell cycle after stimulation by mitogens. The difference is seen after cell treatment with buffers at pH 1.5 (1.3-1.9 range) followed by staining with acridine orange at pH 2.6 (2.3-2.9). Under these conditions the red metachromatic fluorescence of the acridine orange-DNA complex is higher in quiescent cells than in the cycling lymphocytes while the orthochromatic green fluorescence is higher in the cycling, interphase cells. The results suggest that DNA in condensed chromatin of quiescent lymphocytes (as in metaphase chromosomes) is more sensitive to acid-denaturation than DNA in dispersed chromatin of the cycling interphase cells. The phenomenon is used for flow cytometric differentiation between G0 and G1 cells and between G2 and M cells. In contrast to normal lymphocytes the method applied to neoplastic cells indicates the presence of cell subpopulations with condensed chromatin but with DNA content characteristic not only of G1 but also of S and G2 cells. The possibility that these cells represent quiescent (resting) subpopulations, arrested in G1, S and/or G2, is discussed.
Flow cytometric prescreening of cervical smears.
Authors: Linden W A , Ochlich K , Baisch H , Scholz K U , Mauss H J , Stegner H E , Joshi D S , Wu C T , Koprowska I , Nicolini C
Published: April 2011

One-parameter (nuclear DNA) and two-parameter (nuclear DNA and protein or cellular light scatter) measurements of cervical smears were performed using an ICP 11 and a cytofluorograf 4800 respectively. A total of about 1000 cases was analyzed. For the estimation of nuclear DNA alone two fluorochromes were tested (ethidium bromide (EB) and mithramycin (MMC)) combined with three different methods of cell preparation. For the two-parameter measurements cells were double stained with EB and fluorescein isothiocyanate (FITC). Red fluorescence (EB) versus green fluorescence (FITC) or red fluorescence versus scatter were recorded. A computer analysis of the one-parameter histograms was performed using discriminant analysis and the results were compared with the cytodiagnosis of microscopic specimens stained with the Papanicolaou technique. The error rates of the flow cytometric (FCM) data were as follows: (a) standard EB staining, 11% false negative, 26% false positive, 6% unsatisfactory results; (b) pepsination of vital cells and EB staining, 12% false negative, 14% false positive and 4% unsatisfactory results; (c) MMC staining, 10% false negative, 65% false positive and 5% unsatisfactory results. Our two-parameter measurements prove that, as confirmed by cell sorting, red fluorescence versus scatter allows separation of at least three subpopulations in most analyzed samples: (a) anucleated cells; (b) leukocytes; and (c) intermediate and superficial cells.
Quantitative analysis of flow microfluorometric data for screening gynecologic cytology specimens.
Authors: Habbersett M C , Shapiro M , Bunnag B , Nishiya I , Herman C
Published: April 2011

Gynecologic cytology specimens that included the entire spectrum of cervical cytology classification were stained with a combination of propidium iodide and fluorescein isothiocyanate, then analyzed using a flow microfluorometer to measure nucleic acid and protein content, respectively. Numerous descriptors of the resulting two parameter distribution (nucleic acid versus protein) were defined. These descriptors included assessment of the presence or absence of abnormal cells. They also included measures of the staining intensity and dispersion of the normal squamous cell population and the intensity of inflammatory response in the cell population. Relative percentages of inflammatory and epithelial cells were demonstrated to effect the screening performance of this system only in borderline lesions. Decision tree algorithms allowed optimization of the selected parameters for screening logic of normal-abnormal decisions on a specimen-by-specimen basis. In addition, quantitative definitions of specimen adequacy were determined. Appropriate controls for batch staining of specimens were evaluated. These results of applying pattern recognition techniques to flow microfluorometer multiparameter data demonstrate that considerably more information about cell populations and subpopulations can be extracted than heretofore possible.
Cytofluorometric and cytochemical comparisons of normal and abnormal human cells from the female genital tract.
Authors: Gill J E , Wheeless L L , Hanna-Madden C , Marisa R J
Published: April 2011

Acridine orange staining of exfoliated cells from epithelial tissues facilitates discrimination between normal and abnormal cells: abnormal cells develop highly elevated nuclear fluorescence. Comparisons of acridine orange (AO) staining with propidium iodide (PI) or Feulgen staining have shown that: (a) PI staining also provides highly elevated nuclear fluorescence from abnormal cells; (b) the distributions of nuclear fluorescence following AO or PI staining were usually not significantly different as judged by the Kolmogorov-Smirnov test; (c) fluorescence emission spectra from AO and PI stained cells are consistent with the hypothesis that both fluorochromes bind to DNA within cell nuclei; (d) DNAse treatment of AO stained normal cells eliminates the nuclear fluorescence peak from slit-scan contours; RNAse treatment has no effect on nuclear fluorescence; (e) the distribution of abnormal cell nuclear fluorescence after AO staining is usually, but not always, significantly different from the distribution of abnormal cell nuclear absorbance after Feulgen staining, with relative nuclear fluorescence being greater than relative nuclear absorbance. The hypothesis currently most consistent with these results is that elevated Feulgen DNA content can account for only part of the discrimination provided by AO staining, and that the chromatin within abnormal cells is altered so as to increase accessibility of DNA to intercalating dyes.
Pairs of fluorescent dyes as probes of DNA and chromosomes.
Authors: Latt S A , Sahar E , Eisenhard M E
Published: April 2011

If two fluorescent dyes with different binding or fluorescence specificities are used simultaneously to stain DNA or chromosomes, the ratio of their fluorescent signals can provide information about base composition or base analogue substitution. Energy transfer between such dye pairs, possible if the fluorescence spectrum of one overlaps the absorption spectrum of the other, can modify observed fluorescence. Microfluorometric measurements were used to document the occurrence of energy transfer between quinacrine or 33258 Hoechst as energy donor and ethidium or 7-aminoactinomycin D as acceptor when used jointly to stain cytologic preparations of human metaphase chromosomes. Use of 7-aminoactinomycin D, a dye with G-C binding specificity, as energy acceptor permitted the identification of human chromosome regions presumptively enriched for clusters of A-T base pairs, based on the resistance of A-T specific fluorescence, from quinacrine or 33258 Hoechst, to energy transfer dependent quenching. The results provide information about basic structural features of metaphase chromosomes, and the associated methodology may prove useful in accentuating specific fluorescent polymorphic chromosome regions.
Acriflavine-Feulgen stilbene staining: a procedure for automated cervical cytology with a television based system (LEYTAS).
Authors: Tanke H J , van Ingen E M , Ploem J S
Published: April 2011

A sample preparation and staining procedure for automated cytology with a TV based system (LEYTAS) is described. It consists of a centrifugation technique and automated acriflavine-Feulgen stilbene staining of cervical specimens. The advantages of using both the fluorescence and the absorption image of acriflavine-Feulgen stilbene stained cervical cells for a television based system are discussed.
Autoradiographic visualization of beta-adrenergic receptors in normal and denervated skeletal muscle.
Authors: Lavenstein B , Engel W K , Reddy N B , Carroll S
Published: April 2011

Autoradiographic localization of beta-adrenergic receptors in rat skeletal muscle in vivo was achieved utilizing [125I]-iodohydroxybenzylpindolol, a potent beta-adrenergic blocker with high affinity and specificity for those receptors. In normal muscle the beta-adrenergic receptors were localized mainly to blood vessels, arterioles greater than venules, with much less concentration of grains over the fascicles of muscle fibers. One week after denervation there was an increase in binding both to blood vessels and muscle fibers, more so in soleus and gactrocnemius than in extensor digitorum longus. While these results parallel in vitro biochemical studies, they dictate caution when inferring cellular localization of beta-adrenergic receptors (and other molecules) solely on the basis of biochemical techniques applied to subcellular fractions of whole-organ homogenates.
Immunofluorescent localization of pea storage proteins in glycol methacrylate embedded tissue.
Authors: Craig S , Goodchild D J , Millerd A
Published: April 2011

Improved immunofluorescent techniques have been developed for the high resolution light microscopic localization of intracellular antigens in plant tissue. Thin sections of pea cotyledon tissue which had been fixed in paraformaldehyde and embedded in glycol methacrylate were reacted with mono-specific antibodies to the storage proteins legumin and vicilin. These antibodies were raised in sheep, purified by affinity chromatography and tested by immunoelectrophoresis and immunodiffusion. Using the indirect technique, rhodamine-labeled antibodies permitted specific fluorescent localization of the legumin and vicilin to small (ca. 1 micrometer) cytoplasmic organelles in near mature tissue. Subsequent histochemical staining verified the proteinaceous nature of these organelles. Parameters affecting staining specificity and background fluorescence are discussed.
A method for enumeration ot T and B lymphocytes in tissues.
Authors: Bastian F O , Middleditch P R , Bossart M I
Published: April 2011

A method for the identification of T and B lymphocytes in tissue specimens is described. A sonication technique results in viable relatively pure lymphocyte populations which are easily classified by their surface markers. This readily reproducible method can become a standard laboratory procedure in the evaluation of disease states which require such information related to the classification of lymphocyte cell origin.
Somatostatin-immunoreactive nerve cell bodies and fibers in the medulla oblongata et spinalis.
Authors: Forssmann W G , Burnweit C , Shehab T , Triepel J
Published: April 2011

Complete serial sectioning of the medulla oblongata in monkey, cat, guinea pig, and japanese dancing mouse and incubation for somatostatin-immunoreaction was carried out. Numerous regions of the medulla oblongata such as the nucleus reticularis gigantocellularis, nucleus cuneatus et gracillis, nucleus raphe magnus, nucleus tractus solitarius, nucleus vestibularis, and parts of the oliva contain dense networks of somatostatin-immunoreactive nerve fibers. Cell bodies were seen in the nucleus reticularis medullae oblongatae. In the spinal cord the sections from each segment were analyzed, showing the highest concentrations of somatostatinergic fibers in the substantia gelantinosa of the columna dorsalis. Cell bodies were seen in the zona intermedia centralis, especially in the upper cervical segments. Many positive fibers were also seen in the entire zona intermedia and the columna ventralis. Especially prominent was the immunoreactivity in the zona intermediolateralis of the thoracic segments and the columna ventralis of the lower lumbar and sacral segments.
Chemical modification of horseradish peroxidase. Preparation and characterization of tracer enzymes with different isoelectric points.
Authors: Rennke H G , Venkatachalam M A
Published: April 2011

Horseradish peroxidase (HRP), a plant glycoprotein with a molecular weight of 40,000 D and a molecular radius (ae) of 30 A, has been modified chemically to prepare tracer molecules with different molecular charge. Modification of free carboxyl groups on the enzyme is achieved by carbodiimide activation and subsequent reaction of activated carboxyl groups with a nucleophile; uncharged groups or radicals containing additional positively charged moieties are introduced into the protein molecule resulting in an increased net positive charge of the tracer. Amino groups in the protein molecule are modified by acetylation or succinylation; this reaction will increase the net negative charge of the enzyme by either introducing an uncharged group or an additional carboxyl radical. The tracer molecules so obtained are then characterized in terms of molecular size and charge by column chromatography and isoelectric focusing respectively. The enzymatic activity as measured by 3,3'-diaminobenzidine reaction, the pH optimum and the absorption spectra for the modified enzymes remain virtually unchanged.
Pituitary adenomas. Clinico-morphologic correlations.
Authors: Landolt A M
Published: April 2011

Immunohistochemistry and electron microscopy allow new insights into the biology and natural history of the proliferative processes of the hypophysis. Hyperplasia can be diffuse or focal. Its function and/or growth are controlled by hypothalamic factors. Hyperplasia may precede the formation of an adenoma. Function and/or growth of adenomas is partially or completely independent of hypothalamic regulation. Adenomas are divided according to their functional activity into an endocrine active and inactive group. A further subdivision is made according to the secreted hormone. Inactive adenomas may have lost the ability to produce hormones, may secrete hormones at a very low rate, or may secrete abnormal substances.
Myoglobin as an ultrastructural probe for studying permeability of the nephron.
Authors: Anderson W A , Vigil E L
Published: April 2011

Hemeproteins, like cytochrome c (12,500 M.W.; Karnovsky and Rice, 1969) and myoglobin (17,816 M.W.; Anderson, 1972; Simionescu et al., 1973) are advantageous over the true peroxidases with larger molecular weights (e.g. horseradish peroxidase, ca. 40,000 M.W) as ultrastructural probes in that they do not elicit vascular leakage in the inflammatory response (Cotran and Karnovsky, 1967) and are relatively nontoxic immunologically inert substances. The main disadvantage in using cytochrome c and myoglobin is that they have weak peroxidatic activity compared to the true peroxidases (Nakamura et al., 1960; Keilin, 1961; Kurozimi et al., 1961). These hemeproteins, however, offer the following advantages: 1) they retain sufficient peroxidatic activity after aldehyde-fixation to oxidize 3,3'-diaminobenzidine (DAB), 2) they may be localized by virtue of an insoluble reaction product (osmium black) deposited at the site of hemeprotein immobilization by fixation, and 3) they represent low molecular weight probes. This brief report emphasizes the advantages of myoglobin in the study of glomerular permeability, transport by endocytosis in proximal tubules and translocation of protein in the lower segments of the nephron.
Antibody binding by native and denatured myosin and paramyosin.
Authors: Elfvin M J , Levine R J , Pepe F A , Dewey M M
Published: April 2011

By the techniques of immunodiffusion and fluorescent immunohistochemistry we show that antibodies to both the native and the SDS-denatured forms of the proteins, paramyosin and myosin, react with the native, SDS-denatured and glutaraldehyde-fixed forms of their respective antigens. Anti-denatured myosin also binds to both native and denatured forms of the proteolytic subfragments of myosin: globular subfragment-1 and alpha-helical LMM. Anti-native myosin, on the other hand, while able to bind to both native and denatured LMM or rod and to native and glutaraldehyde-fixed S-1, does not bind to SDS-denatured S-1.
Membrane differentiation of developing hemic cells of the bone marrow demonstrated by changes in concanavalin A surface labeling.
Authors: Ackerman G A , Freeman W H
Published: April 2011

The concanavalin A-gold labeled horseradish peroxidase (Con A-HRP-G) method has been employed in the ultrastructural localization of Con A surface receptor sites on glutaraldehyde-fixed normal human and guinea pig bone marrow cells. The number of gold particles per micron of cell surface was counted and data subjected to statistical analysis. All cells of the bone marrow exhibited Con A binding; however, the extent of surface labeling was dependent both on cell type and stage of differentiation. Distinctive modifications in mean surface density correlated with specific periods during the maturation of the erythrocytic, neutrophilic, eosinophilic and monocytic cell series. In several instances, the differentiative changes in surface Con A labeling proved to be species dependent. These observations are discussed in relationship to methodology and to potential changes in number and/or spatial arrangement of Con A receptor sites, primarily attributable to mannosyl and/or glucosyl residues associated with membrane glycoproteins and/or glycolipids of developing neutrophilic and erythrocytic cells.
A fluorescent assay of proteinases in cultured mammalian cells.
Authors: Dolbeare F , Vanderlaan M
Published: April 2011

We have demonstrated proteinase activity in unfixed cells grown on tissue culture plates with a technique using 5-nitrosalicylaldehyde and peptide derivatives of 4-methoxy-2-naphthylamine. The 4-methoxy-2-naphthylamine liberated by proteinase activity reacts with 5-nitrosalicylaldehyde to form a fluorescent product. The substrates CBZ-alanyl-arginyl-arginyl-4-methoxy-2-naphthylamine and lysyl-alanyl-4-methoxy-2-naphthylamine, were used for the direct visual detection of two arylamidase activities in BALB/c 3T3 and C3H 10T 1/2 cells. With low magnification these enzyme activities can be detected in single clones; with higher magnification the fluorescent product can be seen within the cytoplasm of single cells.
A study of acid phosphatase and dipeptidyl aminopeptidase II in monodispersed anterior pituitary cells using flow cytometry and electron microscopy.
Authors: Smith R E , Dean P N
Published: April 2011

A brief historical review of cytoenzymology is presented from the time of introduction into electron microscopy to the present, where the direction for quantification of an enzyme in single cells appears most promising by fluorescent staining. First attempts are reported to quantitate acid phosphatase (AcPase) and dipeptidyl aminopeptidase II (DAP-II) in monodispersed anterior pituitary cells from lactating and postlactating rats by flow cytometry, fluorescent, and electron microscopy. 3-Hydroxy-flavone is introduced as a new fluorescent cytochemical stain for AcPase, useful in flow cytometry but of only limited use in fluorescent microscopy. Histograms for AcPase indicate a single peak of cells staining more intensely in cell preparations from postlactating over lactating animals. Histograms for DAP-II staining indicate two distinct populations of cells present in the lactating and only one in the postlactating rat anterior pituitary gland. The application of dual laser staining indicates that not all cells stain for both enzymes. Electron microscopy shows the subcellular localization of DAP-II to be limited to lytic bodies and in mammotrophic cells to some secretion granules.
Freezing of tissue-limits for the autoradiographic localization of diffusible substances.
Authors: Frederik P M , Busing W M
Published: April 2011

Frozen thin sections and sections from freeze-dried and embedded tissue are used for the autoradiographic localization of diffusible substances at the electron microscope level. The presence of ice crystals in such sections may limit the autoradiographic resolution. Ice crystals are formed during freezing and may grow during subsequent processing of tissue. The contribution of ice crystal growth to the final image was estimated by measuring the distribution of the ice crystal sizes in freeze-etch replicas and in sections from freeze-dried and embedded tissues. A surface layer (10-15 mu) without visible ice crystals was present in both preparations. Beneath this surface layer the diameter of ice crystals increased towards the interior with the same relationship between crystal size and distance from the surface in the freeze-etch preparation as in the freeze-dry preparation. Ice crystal growth occurring during a much longer time during freeze-drying compared to freeze-etching does not significantly contribute to the final image in the electron microscope. The formation of ice crystals during freezing determines to a large extent the image (and therefore the autoradiographic resolution) of freeze-dry preparations and this probably holds also for thin cryosections of which examples are given.
High resolution scintiautoradiography.
Authors: Fischer H A
Published: April 2011

An electron microscopic scintiautoradiographic method for studying histological sections is reported. An increase in the silver grain yield is attained by using a scintillator in embedding medium. The amplification effect for 3H-labeling reaches 3-fold and for 14C reaches a 15-fold. No lessening of autoradiographic resolution was observed. On the basis of these preliminary results only qualitative evaluation of data is discussed.
Numerical density of convex, nonbranching organelles in anisotropically oriented cells. Cilia in tangential chondrocytes.
Authors: Wilsman N J
Published: April 2011

A stereological expression relating the mean number of organelle profiles per cell profile, Np, to the mean number of organelles per cell, Nc, is first derived and then tested for cilia in tangenital articular chondrocytes where Nc is known to be 1.0. Nc derived stereologically for this organelle-cell model is 1.19. This close approximation of the estimate to a known value provides empirical evidence of the usefulness of the derived stereological expression.
Effect of colchicine on the immunohistochemical localization of somatostatin in the rat brain: light and electron microscopic studies.
Authors: Dubé D , Pelletier G
Published: April 2011

Somatostatin (SRIF), the hypothalmic hormone which inhibits the secretion of growth hormone by the pituitary, has been localized immunohistochemically in the rat hypothalamus after intracerebral injection of colchicine. The number of cell bodies staining for SRIF was increased in the periventricular nucleus while the number of nerve fibers was decreased in the median eminence after treatment. The number of secretory granules containing SRIF in the nerve cell bodies was increased in the treated animals, suggesting a correlation between the number of specific secretory granules and intensity of the immunohistochemical reaction. These observations are in agreement with the hypothesis that SRIF cell bodies in the periventricular nucleus send their axons into the median eminence.
Skeletin immunoreactivity in heart Purkinje fibers from several species.
Authors: Eriksson A , Thornell L-E  , Stigbrand T
Published: April 2011

Ever since its discovery, the identification of the specialized conducting system of the heart has been a matter of debate. In some species, a main distinguishing feature under the electron microscope, as compared with ordinary myocytes, is the presence of large pools of juxtanuclear filaments, so called intermediate or skeletin filaments. In the present study, we have adopted the indirect immunofluorescence method and anti-skeletin antisera for the identification of the ventricular conducting system in several species. It was found that anti-skeletin reactivity generally exceeded that of ordinary myocytes. The degree of immunofluorescence could be related to a previous classification model of the differentiation of the conducting cells. It is suggested that skeletin is highly conserved throughout phylogeny and that anti-skeletin may serve as an additional tool for the identification of conducting cells at the light microscopic level.
Fluorescent DNA probes for flow cytometry. Considerations and prospects.
Authors: Crissman H A , Orlicky D J , Kissane R J
Published: April 2011

Techniques employing base specific deoxyribonucleic acid (DNA)-binding fluorochromes and flow cytometry (FCM) are potentially useful for obtaining information of the compositional features of chromatin or chromosomes of mammalian cells. Fluorescent compounds which form complexes preferentially at the A-T rich regions (i.e., DNA-reactive Hoechst dyes) or the G-C rich regions (i.e., mithramycin, chromomycin, olivomycin) in DNA are available and compatible with current FCM technology as are other compounds (i.e., ethidium bromide, propidium iodide) which show little or no base specificity and bind by intercalation in the double stranded regions of helical DNA. Energy transfer between appropriate DNA-bound dyes is a reflection of the quantity and proximity of regions containing the respective base pair segments. Since extrinsic fluorescent probes provide only a measure of available binding sites or regions unobstructed by chromatin-associated or chromosomal-associated proteins, interpretations of fluorescence measurements need to be substantiated by adequate control measures.
Freeze substituted tissue in 5'-nucleotidase histochemistry. Comparative histochemical and biochemical investigations.
Authors: Klaushofer K , von Mayersbach H
Published: April 2011

The suitability of freeze-substitution in n-butanol and paraffin embedding of tissues for the histochemical demonstration of 5'-nucleotidase was investigated and compared with commonly used preparation techniques, such as fresh frozen sections and cryostate sections of cold formalin and glutaraldehyde-fixed rat liver. The influences of each step of the preparation techniques on the enzyme activity were controlled by a quantitative radiochemical assay. Freeze substitution was revealed to be superior to the commonly used preparation techniques with respect to: 1) high sensitivity and specificity of the histochemical 5'-nucleotidase reaction (this is based on the fact that incubation media with very low lead concentrations (0,6 mM/1) can be used); 2) excellent morphological appearance of the tissues showing cytological details of enzyme localization; 3) unlimited storage of the tissue materials and ease of sectioning.
Histochemical comparison of focal losses of RNase and ATPase activities in preneoplastic rat livers.
Authors: Daoust R
Published: April 2011

To better assess the significance of enzyme-deficient foci as putative premalignant lesions, parallel histochemical analyses of RNase and ATPase activities were carried out in serial sections of livers from rats fed 4-dimethylaminoazobenzene. The results showed that focal losses of RNase and canalicular ATPase activities occur simultaneously in congruent areas of liver parenchyma at early stages of carcinogenesis. Such foci presumably represent altered cells capable of progressing to neoplasia since the changes observed in this new cell population persist in developing tumors.
Ferrocyanide staining of transferrin and ferritin-conjugated antibody to transferrin.
Authors: Parmley R T , Ostroy F , Gams R A , DeLucas L
Published: April 2011

To evaluate the ultrastructural distribution of transferrin on the surface of L1210 ascites tumor cells, we used ferrocyanide to stain ferric iron (Prussian blue reaction) in transferrin, as well as in ferritin conjugated to antibody that was immunologically attached to the transferrin. Small deposits averaging 5 nm in diameter identified transferrin iron, whereas large cuboidal deposits averaging 50 nm in diameter stained ferritin conjugated-antibody that was bound to both transferrin and apotransferrin on the cell surface. The ability of transferrin to deliver iron to ascites tumor cells was confirmed by kinetic studies of transferrin labeled with 59Fe and 125I. These preliminary results are consistent with release of transferrin iron at the cell surface and demonstrate additional uses for ferrocyanide in ultrastructural cytochemical techniques.