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Immunofluorescence, confocal microscopy

Key Words Brucella Francisella macrophages intracellular cycle endosomes, endoplasmic reticulum confocal immunofluorescence microscopy digitonin. [Pg.133]

Monitoring Chemokine Receptor Trafficking by Confocal Immunofluorescence Microscopy... [Pg.281]

The cytoplasmic domain of P-selectin contains signals that mediate its rapid en-docytosis in clathrin-coated pits. Interactions of the cytoplasmic domain of P-selectin with these structures were found to enhance its adhesive function under flow [47]. Transfected CHO cells were prepared that express wild-type P-selectin or P-selectin constructs with substitutions or deletions in the cytoplasmic domain that either increase or decrease its internalization rate. Under flow, neutrophils tether equivalently to all constructs when expressed at matched densities. However, neutrophils roll on the internalization-competent constructs with greater adhesive strength, at slower velocities, and with more uniform motion. Confocal immunofluorescence microscopy demonstrates colocalization of a-adaptin, a component of clathrin-coated pits, with wild-type P-selectin but not with internalization-defective P-selectin lacking the cytoplasmic domain. Thus, interactions of P-selectin with clathrin-coated pits provide an alternative to cytoskeletal interactions to enhance adhesive function. The association of P-selectin with clathrin-coated pits may delay dissociation of P-selectin-PSGL-1 bonds and/or prevent forced extraction of P-selectin from the membrane. [Pg.1723]

Since endocytosis ofLDH was confirmed by TEM images (Figure 13.9), forthe next step, its specific endocytic pathway for membrane entry was determined by immunofluorescence and confocal microscopy. Cells were incubated with LDH-FITC, fixed with 3.7% freshly made formaldehyde, and then stained with either anti-clathrin antibody or anti-caveolin-1 antibody both conjugated to the red fluorescent dye Texas Red (TR). The confocal microscopic images showed that green fluorescent... [Pg.413]

An even higher degree of colocalization has been reported in studies using confocal microscopy to detect immunofluorescence for D1 and D2 receptors. These studies have found both types of receptors in virtually all striatal neurons. In cell cultures, D1 and D2 receptors have been colocalized to terminals of intrinsic neurons (Wong et al., 1999). Similarly, Aizman et al. (2000) found that cultured striatal neurons expressed both D1 and D2 receptors. These results are not peculiar to the culture situation. When examined in acutely prepared slices, virtually all cells were positive for both the D1 and D2 subclasses. [Pg.214]

Figure 10.16. Endocytosis of dopamine reuptake transporters triggered by amphetamine, a Application of amphetamine reduces the transmembrane current that is a measure of dopamine reuptake. Shown are several independent experiments, b Detection of the transporter by immunofluorescence and confocal microscopy. In the absence of amphetamine, the transporter is located at the cell surfaces. Amphetamine induces translocation of the fluorescence to intracellular compartments. Figure 10.16. Endocytosis of dopamine reuptake transporters triggered by amphetamine, a Application of amphetamine reduces the transmembrane current that is a measure of dopamine reuptake. Shown are several independent experiments, b Detection of the transporter by immunofluorescence and confocal microscopy. In the absence of amphetamine, the transporter is located at the cell surfaces. Amphetamine induces translocation of the fluorescence to intracellular compartments.
Exposure of DRG neurons to inhibitors of membrane protein internalization (either bafilomycin A or chloroquine) prevented the loss of Na conductance normally observed after HSV-1 infection (Storey et al 1998). Furthermore, confocal microscopy of DRG neurons stained with a pan Na channel antibody showed that HSV-1 infection resulted in a marked loss of plasma membrane staining and an overall reduction in immunofluorescence throughout the neurons (Fig. 5). These findings are consistent with the hypothesis that HSV-1 induces Na channel internalization. A possible mechanism of channel loss is suggested by experiments on veratridine-induced loss of surface Na channels in neonatal (but not adult) rats CNS neurons where a rapid internalization of Na channels (Tiy2 15 min) occurs after a rise in intracellular Na concentration (Dargent Couraud 1990, Dargent et al 1994). HSV-1 induced loss of Na currents in adult rat DRG neurons showed a similar dependence on extracellular Na and was inhibited when extracellular Na was substituted with choline, which raises the possibility that a rise in internal Na can mediate the HSV-1-induced internalization. [Pg.152]

Immunofluorescent detection of TS protein was done with the use of monoclonal antibodies, developed by in vivo immunization of Balb/c mice with homogeneous recombinant rat hepatoma TS protein as an antigen. The specific anti-rat TS antibodies recognized also T. spiralis TS, as indicated by cross-reactivity on Western blot. Localization of the enzyme was based on analysis of pictures collected by confocal microscopy. Two types of T spiralis muscle larvae preparations were studied muscle larvae isolated from mouse muscles by a procedure destroying nurse cells and muscle larvae remaining in nurse cells, isolated as an intact nurse cell preparation. [Pg.334]

Direct immunofluorescence. Rapid simple procedure. Multiple labeling is possible using different filter combinations. Confocal microscopy may be used. Extravagant when more than just a few target antigens are to be studied. [Pg.175]

Fig. 1. Double immunofluorescence detection of cytoplasmic cytokeratin 19 intermediate filaments (CK19) (FITC, green/ filamentous) and the cell-membrane associated connexin 43 (Cx43) gap junction protein (Cy3, red/dot-like) in the stratified squamous epithelium of human cervix. Combination of mouse monoclonal (CK19) and rabbit polyclonal (Cx43) antibodies and distinct fluorochrome-labeled secondary antibodis. Confocal laser scanning microscopy, a projection of five optical layers. Fig. 1. Double immunofluorescence detection of cytoplasmic cytokeratin 19 intermediate filaments (CK19) (FITC, green/ filamentous) and the cell-membrane associated connexin 43 (Cx43) gap junction protein (Cy3, red/dot-like) in the stratified squamous epithelium of human cervix. Combination of mouse monoclonal (CK19) and rabbit polyclonal (Cx43) antibodies and distinct fluorochrome-labeled secondary antibodis. Confocal laser scanning microscopy, a projection of five optical layers.

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Confocal

Confocal microscopy

Confocality

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