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R R Cowden

Publications and source records attributed to R R Cowden.

At least 19 recordsLinked to original sources
PubMed1986

Reactions of seven basic fluorochromes with unfixed cells obtained from the salivary glands of the dipteran fly Megaselia scalaris Loew (Phoridae).

Seven basic fluorochromes with varying specificities were used to stain the large squamous epithelial cells isolated from the larval salivary glands of Megaselia scalaris (Phoridae). Although the EDTA-based method selected for isolating the cells produced permeabilization and a loss of viability of the cells, consistent results were obtained with the various fluorochromes. The "classical" pattern of green nuclear and red cytoplasmic fluorescence observed in cells stained with acridine orange could be changed to green cytoplasmic and red nuclear fluorescence by pretreatment with RNase. The predominantly cytoplasmic and nucleolar fluorescence obtained with pyronine Y could be changed to mainly nuclear fluorescence by RNase pretreatment. The other five fluorochromes tested were not affected appreciably by extraction with RNase. Quinacrine mustard, dicarbocyanine (DiOC3(3)), and rhodamine 123 produced primarily cytoplasmic and nucleolar fluorescence, while nile red revealed mainly cytoplasmic lipid droplets. Phosphine 3R initially stained lipid droplets but very rapidly redistributed throughout the cytoplasm and nucleus. Because of their large size, flatness, and content of histochemically demonstrable components, the cells of Megaselia are especially appropriate for use as "optical objects" or controls in various studies. New methods of isolating the cells, however, will be needed to prevent permeabilization and loss of viability of the cells.

Acridine Orange↗
PubMed1985

Microfluorometric estimates of proteins associated with murine hepatocyte and thymocyte nuclei, residual structures, and nuclear matrix derivatives.

Isolated diploid hepatocyte and thymocyte nuclei and their derivatives ("residual structures" and nuclear matrices, as defined by Kaufmann et al. 1981) were evaluated by microfluorometry following reaction with the following fluorochromes: brilliant sulfaflavine (BSF) used at pH 2.8 for the demonstration of total protein; acridine orange (AO) used at pH 9.0 to reveal acidic groups of proteins; and 3-(4-maleimidylphenyl)-7-diethylamino-4-methylcoumarin (CPM) used under conditions required to demonstrate the sum of sulfhydryl (SH) and disulfide (SS) groups of proteins. The results suggested that the proteins reacting with AO and CPM differed from each other and from those revealed by fluorochroming with BSF. In every comparison, hepatocyte nuclei and their derivatives were more fluorescent than the respective populations of thymocyte nuclei and their derivatives. In material fluorochromed with BSF and AO, nuclear matrices were less fluorescent than residual structures, which, in turn, were less fluorescent than intact nuclei. In contrast, nuclear matrices fluorochromed with CPM were less fluorescent than intact nuclei but more fluorescent (paradoxically) than residual structures. The ratios of the total fluorescence values of hepatocyte and thymocyte nuclei fluorochromed with BSF changed significantly during extractions required to produce residual structures and nuclear matrices, while comparable ratios in material fluorochromed with AO or CPM did not change significantly. Comparisons of the ratios of the fluorescence values of intact nuclei and their derivatives in a variety of combinations yielded complex and variable results.(ABSTRACT TRUNCATED AT 250 WORDS)

Acridine Orange↗
PubMed1983

Evaluation of five basic fluorochromes of potential use in microfluorometric studies of nucleic acids.

Five basic fluorochromes were evaluated to determine whether or not any could be used in microfluorometric studies for the selective demonstration of nucleic acids. Only two of the fluorochromes - Nuclear Yellow (Hoechst 769121) and the phenoxyindole compound D261/37 - were found to be selective for nucleic acids, while the other three fluorochromes produced small to moderate amounts of fluorescence in preparations extracted sequentially with RNase and DNase. All of the fluorochromes could be considered "structural probes" since they produced less fluorescence in the highly condensed chromatin of thymocyte nuclei than they did in the less condensed chromatin of hepatocyte nuclei. When exposed to continuous excitation for 2-min intervals, hepatocyte nuclei stained with Nuclear Yellow or D261/37 gradually lost, respectively, approximately 21% or nearly 60% of their original fluorescence. Nuclei stained with the other three fluorochromes displayed much more rapid fading and lost more than 80% of their original fluorescence.

Animals↗
PubMed1981

Microfluorometric investigations of chromatin structure. I. Evaluation of nine DNA-specific fluorochromes as probes of chromatin organization.

The ability of the highly condensed chromatin of small thymocyte nuclei and the more loosely organized chromatin of hepatocyte nuclei to interact with nine DNA-specific fluorochromes was assessed by microfluorometry. Although the results obtained with five of the fluorochromes - mithramycin, 7-aminoactinomycin D, Hoechst 33258, DAPI, and propidium iodide - were found to be virtually unaffected by differences in the degree of condensation of the chromatin, the values obtained with the remaining fluorochromes - proflavine, quinacrine mustard, berberine sulfate, and pyronin Y - appeared to be affected significantly by organizational differences of the chromatin. All of the latter "structural probes," except quinacrine mustard, produced fluorescence values which were higher in the 2c nuclei of hepatocytes than in the nuclei of small thymocytes. Quinacrine mustard yielded higher values in thymocyte nuclei; and in the hepatocyte polyploid series (2, 4, and 8c), it did not produce the expected multiples of the 2c value. Pretreatment of the two types of nuclei with RNase affected their total fluorescence in unpredictable ways. While RNase extraction lessened the differences between thymocyte and 2c hepatocyte nuclei stained with propidium iodide, Hoechst 33258, proflavine, and berberine sulfate, it increased the differences between nuclei stained with mithramycin, quinacrine mustard, pyronin Y, and 7-aminoactinomycin D. The ability of RNA-depleted chromatin to interact with various types of fluorochromes might be a useful parameter in subsequent studies of chromatin organization.

Animals↗
PubMed1981

Four fluorochromes for the demonstration and microfluorometric estimation of RNA.

Microfluorometric estimates of total RNA were made in selected test material stained with berberine sulfate, acridine orange, and Hoechst 33258. These measurements were compared with those obtained with propidium iodide, which is known to interact only with double-stranded nucleic acids. It was observed that all of the fluorochromes, including propidium iodide, yielded very similar patterns of fluorescence in the various types of material tested. In isolated thymocyte and hepatocyte nuclei stained with either propidium iodide or Hoechst 33258 at pH 2, it was evident that RNA could be estimated only indirectly by measuring the amount of fluorescence before and after extraction with RNase. It was apparent that the total fluorescence of small thymocyte nuclei was affected much less by RNase extraction than that of 2c hepatocyte nuclei. Attempts to obtain direct estimates of RNA by exposing the preparations to DNase were not successful: the fluorescence of thymocyte nuclei dropped almost to zero, and hepatocyte nuclei could no longer be assigned to distinct ploidy classes. In addition, since the highly condensed chromatin of thymocyte nuclei was stained much more prominently than the looser chromatin of hepatocyte nuclei with Hoechst 33258, it was apparent that this fluorochrome - when used at pH 2 - has potential usefulness as a "probe" of organizational differences in chromatin.

Acridine Orange↗
PubMed1980

Ultrastructural and gas-chromatographic analysis of the preputial glands of male nude (nu/nu) mice.

The preputial glands of male nude (nu/nu) mice were analyzed by a combination of electron microscopy and gas chromatography to determine whether or not they are affected, like developing hairs and nails, by the nu/nu genotype. Results of the analyses revealed no differences between the glands of nude and normal male mice in either their ultrastructural characteristics or lipid secretory products.

Animals↗
PubMed1980

Demonstration of sulfhydryl and disulfide groups by a fluorescent maleimide procedure.

Several fluorescent maleimide compounds were evaluated as possible substitutes for N-(4-aminophenyl)maleimide in the histochemical procedures developed by Sippel (1973, 1978a, b, 1980) for the demonstration of sulfhydryl and disulfide groups. The brightest and most selective fluorescence was obtained by using N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM), although both eosin-5-maleimide and fluorescein-5-maleimide could also be used if adequate control preparations were made.

Animals↗
PubMed1980

Effects of preparation and fixation on three quantitative fluorescent cytochemical procedures.

A series of experiments was undertaken in which cells dissociated from the abdominal lymph nodes of mice were lightly centrifuged into slides and fixed either wet or after drying in 70% ethanol, 1% glutaraldehyde, 1% formaldehyde, or neutral formalin. Three fluorescent cytochemical methods were evaluated: staining of DNA with mithramycin; fluorochroming of basic groups of proteins with brilliant sulfaflavine (BSF); and staining of sulfhydryl and disulfide groups with N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM). In the case of mithramycin, the best results were obtained after fixation in 70% ethanol without drying. Staining of dried preparations fixed in 1% glutaraldehyde also yielded reasonably consistent results, although the fluorescence was lower, and the variability higher, than in the group fixed without drying in 70% ethanol. The use of fixatives containing formaldehyde resulted in fluorescence values of only about one-third those of the other two groups, and the variability of the data was higher. In material stained with BSF, satisfactory results were obtained in preparations fixed without drying in neutral formalin containing mersalyl acid. Other fixatives could be used, but the resulting coefficients of variation were higher than those of formalin-fixed material. Sulfhydryl to disulfide ratios approaching those expected from biochemical evidence were obtained in DACM-stained material only after fixation without drying in neutral formalin containing mersalyl acid. Inverted sulfhydryl-disulfide ratios were observed in material fixed without drying in 70% ethanol; and in dried material fixed in 1% formaldehyde, neutral formalin, or 1% glutaraldehyde.

Animals↗
PubMed1979

Ultrastructural and histochemical features of the thymus glands of the adult lungless salamander, Plethodon glutinosus (Caudata: Plethodontidae).

The thymus glands of adult slimy salamanders (Plethodon glutinosus) were examined by light and electron microscopy with the objective of describing the populations of epithelial cells believed to be secretory. The results of various histochemical procedures designed to demonstrate nucleic acids, proteins, lipids, and mucosubstances were evaluated by light microscopy. Each thymus is incompletely subdivided into a variable number of interconnected lobules by trabeculae extending inward from a thin capsule composed of connective tissues. The thymic parenchyma lacks distinct cortical and medullary regions, although developing lymphocytes and plasma cells tend to accumulate in larger numbers in the outermost portions of the glands. Basophils are found regularly in the capsule and trabeculae, but only very rarely within the thymic parenchyma. The epithelial cells of the thymus can be classified into five categories: epithelial reticular cells; three varieties of granulated cells (types I, II, and III), and myoid cells. Epithelial reticular cells form a three-dimensional network which extends throughout all portions of the thymus. Type I and type II granulated cells can be distinguished from one another by various morphological criteria at the ultrastructural level, but only small differences in the composition of their inclusions can be demonstrated histochemically. Both types of granules are composed principally of a proteinaceous material containing an abundance of primary amino and guanidyl groups. In addition, most type I inclusions possess a lipid component that cannot be demonstrated in type II granules. Type III granulated cells possess very small cytoplasmic inclusions resembling those of gastroenteric endocrine cells. Myoid cells contain concentrically arranged myofibrils composed of sarcomeres. In favorably oriented material, small cysts can be identified whose walls are composed of mixtures of type I cells, type II cells, and epithelial reticular cells. Groups of degenerating epithelial cells form lamellated structures corresponding to Hassall's (thymic) corpuscles.

Animals↗