texas-red and lucifer-yellow

Within the corneal stroma, keratocytes communicate through gap junctions. These plasma membrane channels, which connect the cytoplasm of adjacent cells, are composed of connexins. In a cell culture model, an investigation was conducted to determine whether connexin-based gap junction intercellular communication is present in fibroblasts and myofibroblasts, both of which replace keratocytes after wounding.. Fibroblasts and myofibroblasts were grown according to preestablished methods. Phenotype was determined by immunocytochemistry. A gap junction-permeant dye, Lucifer yellow or Cascade blue, and nonpermeant 10-kDa Texas red-dextran were used. Tracer fluorescent dyes were introduced by scrape-loading or by microinjection, and their diffusion into adjacent cells was recorded photographically. Inhibition of gap junction dye transfer was elicited by treatment with 18-alpha-glycyrrhetinic acid (AGA).. In confluent fibroblast or myofibroblast cultures, the scrape-loaded dextran probe remained within wounded cells, whereas the Lucifer yellow or Cascade blue dye diffused into adjacent intact cells. Similarly, in nonconfluent fibroblast and myofibroblast cultures, microinjected Lucifer yellow rapidly diffused from the microinjected cell to adjacent cells. Treatment with 2 microM AGA, an uncoupling agent, blocked the spread of Lucifer yellow in fibroblast and myofibroblast cultures.. Cultured fibroblasts and myofibroblasts have functional gap junctions as has previously been demonstrated for keratocytes in vivo. Thus, fibroblasts and myofibroblasts have the ability to establish and maintain intercellular communication with themselves and with nonactivated keratocytes. This property may be critical in the wound-healing process, especially in the avascular corneal environment.

Topics: Actins; Animals; Cell Communication; Cell Membrane Permeability; Cells, Cultured; Corneal Stroma; Fibroblasts; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Gap Junctions; Glycyrrhetinic Acid; Isoquinolines; Microinjections; Organometallic Compounds; Organophosphorus Compounds; Rabbits; Xanthenes

A method for visualization of individual human brain cells and their dendritic extensions in combination with immunofluorescence is described. Microinjection of Lucifer Yellow was used to reveal the dendritic morphology of cortical brain cells. Indirect immunofluorescence with Texas Red as label was used to investigate the distribution of 3 different groups of immunogens: enzymes (monoamine oxidase A and B), receptors (beta-adrenoceptor protein), and synaptic vesicle proteins (synapsin I and synaptophysin) in each cortical slice. A dual-channel confocal laser scanning microscope with an argon/krypton laser was used for imaging these double-stained fluorescent specimens. Lucifer Yellow and Texas Red were recorded simultaneously or separately, taking advantage of the different activating lines (488 lambda and 568 lambda) of the laser and using the two filter blocks (K1 and K2) supplied with the instrument (BioRad MRC-600) for recording the emission of either fluorophore. Using this technique we have demonstrated the localization of immunoreactive material in relation to the dendritic morphology of cortical cells.

Topics: Brain; Child, Preschool; Female; Fluorescent Antibody Technique; Fluorescent Dyes; Humans; Isoenzymes; Isoquinolines; Microinjections; Microscopy, Confocal; Monoamine Oxidase; Nerve Tissue Proteins; Pilot Projects; Receptors, Adrenergic, beta; Synaptic Vesicles; Xanthenes

A rapid, simple assay for aldehydes generated by oxidation of saccharide units in glycoproteins, using dyes containing hydrazide functionalities, is described. The assay is used, in conjunction with tests of biological activity, to predict oxidation conditions that will result in a maximum of active protein coupled to a hydrazide chromatographic support. Glycoproteins are labeled with Lucifer Yellow CH or Texas Red Hydrazide, and the extent of labeling is determined. Using the assay, it is shown that the efficiency of coupling to Affi-Prep Hydrazide is proportional to oxidation.

Topics: Aldehydes; Animals; Cattle; Chromatography; gamma-Globulins; Glycoproteins; Horseradish Peroxidase; Humans; Isoquinolines; Oxidation-Reduction; Serum Albumin, Bovine; Xanthenes

We have examined five conjugated 10,000 mol. wt. dextrans as potential anterograde tract tracers: Lucifer Yellow, Texas Red, fluorescein, Cascade Blue and tetramethylrhodamine. Pressure injections were made into the brain, dorsal root ganglia or footpads of adult rats. The retrograde tracer Fluoro-Gold was injected alone or mixed with the dextrans before injection. Three-14 days after injection, animals were perfused and sections cut with a freezing microtome. Texas Red-, fluorescein- and tetramethylrhodamine-conjugated dextrans produced intense labeling of neuronal cell bodies, axons and dendritic processes at the injection site and were transported by neurons predominantly in an anterograde direction to yield terminal and preterminal labeling. Relative to the fluorescein and tetramethylrhodamine conjugates, the quality and intensity of the anterograde labeling produced by Texas Red was variable. Results with Lucifer Yellow and Cascade Blue conjugates were negative. Optimal results were produced by slow-pressure injections via glass micropipettes. In comparison with Fluoro-Gold, retrograde transport by the dextran conjugates was present, but limited in its extent. Injections of the tetramethylrhodamine conjugate into dorsal root ganglia produced anterograde labeling of afferent fibers in visceral organs and injections into the nucleus ambiguous labeled motor fibers in the esophagus. Double/triple labeling was observed in the brain and spinal cord following multiple injections of fluorescein, tetramethylrhodamine and Fluoro-Gold. Also, Fluoro-Gold could be mixed with one of the dextrans in order to produce specific retrograde and anterograde labeling from the same injection site. The conjugates were compatible with fluorescent immunocytochemical procedures, but proved unsuitable for peripheral injections.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Brain; Dextrans; Female; Fluorescein; Fluoresceins; Fluorescent Dyes; Hypothalamus; Immunohistochemistry; Isoquinolines; Male; Phytohemagglutinins; Rats; Rats, Inbred Strains; Rhodamines; Staining and Labeling; Stereotaxic Techniques; Stilbamidines; Xanthenes

In the cerebral cortex, the plant lectin Vicia villosa (VVA) selectively stains the surfaces of nonpyramidal neurons. This lectin binds specifically to alpha- and beta-linked N-acetylgalactosamine (GalNac). VVA-reactive carbohydrate is highly concentrated in layer 4 of the primary visual cortex of the cat, where it is associated exclusively with GABAergic local circuit neurons. We have studied this neuronal subset with intracellular electrophysiological recording and dye marking to identify the particular cell types expressing surface GalNac. Five different types of local circuit neurons were stained intracellularly (N = 45), but only 2 types, the columnar basket and large neurogliaform cells, were also labeled by the lectin (N = 19/45). Lectin negative types included small basket, chandelier, and large bitufted cells (26/45). Spiny stellate and pyramidal neurons were also lectin negative. Electrophysiological recordings revealed differences in the duration of action potentials in smooth versus spiny stellates but no differences between lectin-positive or -negative types. A biochemical analysis of cortical glycoproteins by SDS-PAGE and lectin blotting revealed multiple bands containing GalNac enriched in membrane fractions. These carbohydrate-containing molecules may be part of a biochemical mechanism for linking together cells with common functional properties.

Topics: Acetylgalactosamine; Animals; Axons; Cats; Cell Membrane; Chemical Phenomena; Chemistry; Electrophysiology; Fluorescent Dyes; Galactosamine; Glycoconjugates; Glycoproteins; Isoquinolines; Lectins; Microscopy, Fluorescence; Neuroglia; Neurons; Plant Lectins; Visual Cortex; Xanthenes

The bright red fluorescent dye, Texas red, is introduced for ionophoretic microinjection in conjunction with the well-known dye Lucifer yellow. Different identified neurones can thus be visualised in two strongly contrasting colours in the same preparation (differential intracellular staining) following their physiological characterisation. Satisfactory results were obtained with electrodes filled with 4% Texas red (sulforhodamine 101 acid chloride; w/v) in 1 M potassium acetate (pH 3.0) and 5% Lucifer yellow (w/v) in aqua dest., respectively. Texas red was injected ionophoretically with pulsed depolarising current (3-10 nA, 500 ms pulses at 1 Hz, 15-30 min) and Lucifer yellow with hyperpolarizing constant current (5-6 nA, 5-15 min). Histological tissue processing was identical for both dyes, the quality of intracellular recordings with Texas red electrodes was similar to that with Lucifer yellow electrodes. Stained neurones could be visualised in both whole-mounts and sectioned preparations. Differential staining of two identified synaptically coupled neurones, a motoneurone and an interneurone, in the mesothorax of Locusta is presented as an illustration for the possible localisation of contact sites at the light-microscopic level.

Topics: Animals; Female; Fluorescent Dyes; Grasshoppers; Interneurons; Isoquinolines; Male; Motor Neurons; Xanthenes

In this paper, we have characterized the apical and basolateral endocytic pathways of epithelial MDCK cells grown on filters. The three-dimensional organization of the endocytic compartments was analyzed by confocal microscopy after internalization of a fluorescent fluid-phase marker from either side of the cell layer. After 5 min of internalization, distinct sets of apical and basolateral early endosomes were observed lining the plasma membrane domain from which internalization had occurred. At later time points, the apical and the basolateral endocytic pathways were shown to converge in the perinuclear region. Mixing of two different fluorescent markers could be detected after their simultaneous internalization from opposite sides of the cell layer. The extent of the meeting was quantitated by measuring the amount of complex formed intracellularly between avidin internalized from the apical side and biotinylated horseradish peroxidase (HRP) from the basolateral side. After 15 min, 14% of the avidin marker was complexed with the biotinylated HRP and this value increased to 50% during a subsequent chase of 60 min in avidin-free medium. We also determined the kinetics of fluid internalization, recycling, transcytosis, and intracellular retention using HRP as a marker. Fluid was internalized with the same rates from either surface domain (1.2 x 10(-4) microns 3/min per microns 2 of surface area). However, significant differences were observed for each pathway in the amounts and kinetics of marker recycled and transcytosed. The content of apical early endosomes was primarily recycled and transcytosed (45% along Bach route after 1 h internalization), whereas delivery to late endocytic compartments was favored from the basolateral early endosome (77% after 1 h). Our results demonstrate that early apical and basolateral endosomes are functionally and topologically distinct, but that the endocytic pathways converge at later stages in the perinuclear region of the cell.

Topics: Animals; Antibodies, Monoclonal; Cell Compartmentation; Cell Line; Dogs; Endocytosis; Epithelium; Fluorescent Dyes; Image Processing, Computer-Assisted; Immunoenzyme Techniques; Intracellular Fluid; Isoquinolines; Kidney; Microscopy, Fluorescence; Xanthenes

We have used cell fusion to address the question of whether macromolecules are rapidly exchanged between lysosomes. Donor cell lysosomes were labeled by the long-term internalization of the fluid-phase pinocytic markers, invertase (sucrase), Lucifer Yellow, FITC-conjugated dextran, or Texas red-conjugated dextran. Recipient cells contained lysosomes swollen by long-term internalization of dilute sucrose or marked by an overnight FITC-dextran uptake. Cells were incubated for 1 or 2 h in marker-free media before cell fusion to clear any marker from an endosomal compartment. Recipient cells were infected with vesicular stomatitis virus as a fusogen. Donor and recipient cells were co-cultured for 1 or 2 h and then fused by a brief exposure to pH 5. In all cases, extensive exchange of content between donor and recipient cell lysosomes was observed at 37 degrees C. Incubation of cell syncytia at 17 degrees C blocked lysosome/lysosome exchange, although a "priming" process(es) appeared to occur at 17 degrees C. The kinetics of lysosome/lysosome exchange in fusions between cells containing invertase-positive lysosomes and sucrose-positive lysosomes indicated that lysosome/lysosome exchange was as rapid, if not more rapid, than endosome/lysosome exchange. These experiments suggest that in vivo the lysosome is a rapidly intermixing organellar compartment.

Topics: Acid Phosphatase; Animals; beta-Fructofuranosidase; Cell Fusion; Cell Line; Cricetinae; Cricetulus; Dextrans; Female; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescent Dyes; Glycoside Hydrolases; Histocytochemistry; Isoquinolines; Kinetics; Lysosomes; Ovary; Pinocytosis; Xanthenes

We have developed a new two-color fluorescent method for the quantitative measurement of adherence and ingestion of Streptococcus pneumoniae by human monocytes. The method employs a fluorescent naphthalimide, Lucifer Yellow VS, that has been covalently linked to the bacterial cell wall. Bacteria were opsonized and allowed to adhere to monocytes. Lucifer Yellow did not alter the bacterial interaction with complement in serum or with the phagocytic cell. The ability of monocytes to ingest the adherent bacteria was tested under a variety of conditions. Rabbit antibody to Lucifer Yellow derivatized with Texas Red was used to detect monocyte-bound, but uningested bacteria. Dual laser flow cytometry simultaneously quantitated the total number of monocyte-associated S. pneumoniae and the number that remained surface adherent. This method allows separate analysis of the opsonins and receptors involved in bacterial adherence to phagocytes and in the ingestion process.

Topics: Adhesiveness; Antibodies, Bacterial; Cytochalasin B; Humans; Hydrogen-Ion Concentration; Isoquinolines; Monocytes; Opsonin Proteins; Phagocytosis; Streptococcus pneumoniae; Temperature; Time Factors; Xanthenes