Journal: PLoS ONE

Article Title: Microglia Actively Regulate the Number of Functional Synapses

doi: 10.1371/journal.pone.0056293

Figure Lengend Snippet: Microglia near neurons in co-cultures engulf neuronal materials. A. Hippocampal neurons 19 DIV were coincubated with microglia for 2 days, immunostained with CD11b (green), a marker of microglia, and GluA1 (red), and visualized with Alexa Fluor488 or 555-conjugated secondary antibodies. Scale bar, 20 µm. B. Images showing microglia (phalloidin, red) and neurons (obtained from β-actin-EGFP mice, green) in co-cultures. Arrows point to neuronal material within the microglia. Scale bars, 20 µm (left); 5 µm (right). C. Images showing Mac-2 + microglia (green), the lysosomal marker LAMP-1 (red), and presynaptic puncta immunostained for synapsin I (white) in co-culture. Scale bars, 20 m. D. Orthogonal views of the engulfed presynaptic material (white arrowheads in C ).

Article Snippet: The slices and the cultures were incubated with Alexa Fluor488-, Alexa Fluor555- (Invitrogen), or Dylight 649- (Jackson Immunoresearch) conjugated secondary antibodies for 2 h at room temperature to enable fluorescent detection.

Techniques: Marker, Mouse Assay, Co-Culture Assay

Journal: PLoS ONE

Article Title: Microglia Actively Regulate the Number of Functional Synapses

doi: 10.1371/journal.pone.0056293

Figure Lengend Snippet: Microglia alter synaptic density and GluA1 expression in hippocampal neurons. A, B. Hippocampal slices were treated with clodronate (5 µg/ml) or MIF (100 µg/ml) for 6 or 14 DIV. A, GluA1 expression in the CA1 neuronal layer in DIV6 and DIV14 hippocampal organotypic brain slices. B, Lysates from the organotypic slices were immunoblotted and probed using antibodies against GluA1 and α-tubulin. Scale bar, 10 µm. C, D. Primary WT hippocampal neuronal cultures were cultured in the absence or presence of microglia for 2 days. C, Immunohistochemistry was performed using anti-GluA1 antibody under non-permeabilized staining conditions and visualized with Alexa Fluor488-conjugated secondary antibody. After subsequent permeabilization phalloidin was used to stain F-actin. Scale bar, 20 µm. D, Quantitative analysis of surface GluA1 along dendrites for neurons with or without microglia using the LSM 5 Image Browser (Zeiss). Data are presented as mean ±SEM and expressed as a percent of the neurons-only control sample. E–H. E, Hippocampal neurons with or without microglia were stained with PSD95 (green), synapsin I (blue), and phalloidin (red). The smaller boxes show magnified images. Arrows depict PSD95 + synapsin I − puncta. Scale bars, 20 µm (upper panel); 5 µm (lower panel). Quantification of spine numbers ( F ), PSD95 + synapsin I + puncta ( G ), and PSD95 + synapsin I + puncta in total PSD95 + puncta ( H ) in neurons cultured with or without microglia. Values are presented as mean ±SEM expressed as a percent of the neurons-only control sample.

Article Snippet: The slices and the cultures were incubated with Alexa Fluor488-, Alexa Fluor555- (Invitrogen), or Dylight 649- (Jackson Immunoresearch) conjugated secondary antibodies for 2 h at room temperature to enable fluorescent detection.

Techniques: Expressing, Cell Culture, Immunohistochemistry, Staining

Journal: The Journal of Neuroscience

Article Title: Delayed Maturation of Fast-Spiking Interneurons Is Rectified by Activation of the TrkB Receptor in the Mouse Model of Fragile X Syndrome

doi: 10.1523/JNEUROSCI.2893-16.2017

Figure Lengend Snippet: Synapse density and intracortical evoked EPSCs are reduced in Fmr1 KO mice. A , Layer IV interneurons filled with biocytin and tagged with streptavidin-conjugated AlexaFluor 488. Slice containing labeled cell was embedded in LR White resin and trimmed for ultrathin cutting of 70-nm-thick sections. Scale bar, 200 μm. B , Seventy-nanometer-thick serial sections through a segment of dendrite with example of inclusion and exclusion synaptic analysis. Interneuron dendrite (green), PSD-95 (red), and synaptophysin (blue). White and red circles indicate counted and excluded synapses, respectively. To be counted as a synapse, PSD-95 puncta (postsynaptic marker) must overlap with the dendrite on consecutive sections and lie within 100 nm of synaptophysin puncta (presynaptic). Scale bar, 1 μm. C , Three-dimensional renderings of dendritic segments showing synaptic puncta on dendrite from Fmr1 WT and ( D ) Fmr1 KO mice. PSD-95 is labeled red and synaptophysin blue. Scale bar, 1 μm. E , Grouped data for analysis of synapse density in FS interneurons from Fmr1 WT and Fmr1 KO mice. The density of labeled puncta was significantly lower in Fmr1 KO mice (WT: 0.64 ± 0.15 synapses per μm, n = 13 cells from 3 mice; KO: 0.25 ± 0.08 synapses per μm, n = 15 cells from 3 mice, p

Article Snippet: Recorded neurons in slices were labeled with AlexaFluor 488 streptavidin (Life Technologies), 1:200 dilution, and processed as previously described ( ).

Techniques: Mouse Assay, Labeling, Marker

Journal: Biochimica et biophysica acta

Article Title: Aβ truncated species: Implications for brain clearance mechanisms and amyloid plaque deposition

doi: 10.1016/j.bbadis.2017.07.005

Figure Lengend Snippet: Immunofluorescence analysis of Aβ deposits in Alzheimer’s disease specimens Cortical tissue sections were immunostained with 4G8/6E10 in combination with either anti-Aβ4-x or anti-Aβx-34 antibodies (a–f) . Co- localization of the N- and C-terminal truncated species with fibrillar structures was assessed by counterstaining the sections with thioflavin-S (g–l) . (a and d) illustrate reactivity with 4G8/6E10 monoclonal anti-Aβ antibodies followed by Alexafluor-568 conjugated secondary antibody (red signal); (b , g) immunoreaction with rabbit polyclonal anti-Aβ4-x followed by detection with Alexafluor-488 and -568 conjugates (green and red fluorescence, respectively); (e , j) immunoreaction with rabbit polyclonal anti-Aβx-34 and subsequent detection with either Alexafluor-488 or -568 conjugates, respectively; (h , k) thioflavin-S counterstaining. In all cases, right panels (c, f, i , and l) illustrate merged images of the respective stainings. Bar represents 100μm in panels ( a-f ) and 50 μm in panels ( g-l )

Article Snippet: These steps were followed by incubation with the corresponding secondary antibodies labeled with Alexafluor-488 and -568 conjugates (Life Technologies, 1:200, 30 min, RT).

Techniques: Immunofluorescence, Fluorescence

Journal: The Journal of Neuroscience

Article Title: CD44 Signaling Mediates High Molecular Weight Hyaluronan-Induced Antihyperalgesia

doi: 10.1523/JNEUROSCI.2695-17.2017

Figure Lengend Snippet: In vitro LMWH, but not HMWH, increases DRG neuron excitability. A , Control group. B , LMWH group. C , HMWH group. Left columns: Images of neurons, obtained by transmitted light (differential interference contrast, left images) or fluorescence microscopy (right images, in which the darker regions correspond to more intense IB4 binding). A , B , IB4 + neurons stained with IB4 conjugated with AlexaFluor-488 dye. C , The lack of staining determines the IB4 − neuron. Arrows indicate the neurons from which the electrophysiological recordings of APs (middle and right columns) were obtained. A–C , Middle columns: The two sets of traces represent AP generation before (left) and after (right) intervention. Black traces represent AP generation in response to rheobase current injection. Gray traces represent the responses to stimulation below rheobase (no AP generation). The magnitude of current pulses is indicated above the inset boxes (gray). Right column: Traces represent the AHP development and recovery after AP induced by 1 ms current pulse before (black traces) and after (gray traces) intervention. Gray boxes represent stimulation profile in current-clamp mode. D , Pooled relative changes of AP parameters after application of perfusion solution alone (white bars), LMWH (black bars), or HMWH (gray bars). Rheobase (left) was significantly decreased in the LMWH group compared with the control and HMWH groups ( F (2,28) = 11, p = 0.0003, one-way ANOVA, followed by Bonferroni post hoc test; t (22) = 3.5, ** p

Article Snippet: Cells were incubated in Tyrode's solution supplemented with 10 μg/ml IB4 conjugated to AlexaFluor-488 dye (Invitrogen) for 10–12 min in the dark.

Techniques: In Vitro, Fluorescence, Microscopy, Binding Assay, Staining, Injection, Mass Spectrometry

Journal: PLoS ONE

Article Title: A Genetic Screen for Mutations Affecting Cell Division in the Arabidopsis thaliana Embryo Identifies Seven Loci Required for Cytokinesis

doi: 10.1371/journal.pone.0146492

Figure Lengend Snippet: Morphological and ultra-structural characteristics of tio and rsw mutants. Left: Seedling morphology. Images of wild type ( a ) tio-12 ( d ) and rsw-lph ( e ) seedlings; root hairs were contrasted with methylene blue; scale bar equals 1 mm. Center: Anatomy of embryos. Confocal micrographs of wild type ( b ), tio-12 ( e ), and rsw-lph ( h ) embryos at the heart stage of development stained with Alexafluor 488 hydrazide; scale bar equals 30 μm. Right: Ultra-structure of embryonic cells. Transmission electron micrographs showing cells of wild type ( c ), tio-10 ( f ) and rsw-lph ( i ) embryos; the arrowheads point to cell wall stubs, and the stars in ( c ) and ( f ) mark the nucleus; the side of panels equals 10 μm.

Article Snippet: Briefly, embryos were dissected from the seed, collected in 70% ethanol, extracted in 1:1 chloroform/methanol for 30 minutes and 100% methanol for 15 minutes, equilibrated in buffer (50 mM sodium phosphate, 0.05% Triton x-100, pH 7.2) and stained with Alexafluor 488 Hydrazide for two hours in the dark (Molecular Probes; 150 ug/ml in buffer).

Techniques: Staining, Transmission Assay

Journal: PLoS Pathogens

Article Title: Actin Dynamics Regulate Multiple Endosomal Steps during Kaposi's Sarcoma-Associated Herpesvirus Entry and Trafficking in Endothelial Cells

doi: 10.1371/journal.ppat.1000512

Figure Lengend Snippet: Association of Alexafluor 488-transferrin with actin filaments at different stages of endocytosis in endothelial cells. (A–D) HUVEC were treated with Alexafluor 488-transferrin for 60 min, fixed and stained for actin filaments (red), markers of endosomes (white) and nuclei (blue). Alexafluor 488-transferrin shown in green was clearly associated with actin filaments and endosomal protein EEA1 (A), Rab11 (B), Rab7 (C) and LAMP-1 (D). The sections highlighted by the squares are enlarged in the lower or right panels. For corresponding 3D projection of colocalization of Alexafluor 488-transferrin with actin filaments and EEA1, Rab11, Rab7 and LAMP-1, see Videos S16 , S17 , S18 , S19 and S20 . (E–F) Quantification of colocalization of Alexafluor 488-transferrin with markers of endosomes (E), and with both actin filaments and markers of endosomes (F).

Article Snippet: AlexaFluor 488-phalloidin, AlexaFluor 488-transferrin, AlexaFluor 488-CTB, secondary antibodies AlexaFluor 568 goat-anti-mouse IgG1, AlexaFluor 568 and 647 goat anti-mouse IgG2a, and AlexaFluor 488 goat anti-rabbit IgG were from Molecular Probes, Invitrogen (Carlsbad, CA).

Techniques: Staining

Journal: PLoS Pathogens

Article Title: Actin Dynamics Regulate Multiple Endosomal Steps during Kaposi's Sarcoma-Associated Herpesvirus Entry and Trafficking in Endothelial Cells

doi: 10.1371/journal.ppat.1000512

Figure Lengend Snippet: Inhibition of endosomal acidification reduces the entry and trafficking of KSHV particles to the perinuclear region. (A, D, and G) HUVEC were treated with chemical inhibitors of endosomal acidification at indicated doses for 1 h prior to inoculation with KSHV. Cells were inoculated with KSHV in the presence of inhibitors, fixed at 8 hpi, and stained for Orf65+ viral particles (red) and nuclei (blue). (B, E and H) The total number of nuclei bearing at least one Orf65+ particle was determined and calculated as Orf65+ nuclei (%). In parallel experiments, cells were subjected to the same treatments and evaluated for viability by PI staining. (C, F and I) The total number of Orf65+ particles docked at each nucleus was determined. (J) Inhibition of endosomal acidification reduces the internalization of AlexaFluor 488-transferrin. HUVEC were pretreated with inhibitors of clathrin assembly for 1 h prior to exposure to AlexaFluor 488-transferrin. The reduced internalization and perinuclear accumulation of transferrin (green) compared to untreated control cells demonstrates the effective inhibition of endosomal acidification.

Article Snippet: AlexaFluor 488-phalloidin, AlexaFluor 488-transferrin, AlexaFluor 488-CTB, secondary antibodies AlexaFluor 568 goat-anti-mouse IgG1, AlexaFluor 568 and 647 goat anti-mouse IgG2a, and AlexaFluor 488 goat anti-rabbit IgG were from Molecular Probes, Invitrogen (Carlsbad, CA).

Techniques: Inhibition, Staining

Journal: PLoS Pathogens

Article Title: Actin Dynamics Regulate Multiple Endosomal Steps during Kaposi's Sarcoma-Associated Herpesvirus Entry and Trafficking in Endothelial Cells

doi: 10.1371/journal.ppat.1000512

Figure Lengend Snippet: Disruption of actin dynamics reduces the entry and trafficking of KSHV particles to the perinuclear region. (A, E and I) HUVEC were treated with chemicals to disrupt actin dynamics at the indicated doses for 1 h. Cells were inoculated with KSHV in the presence of inhibitors, fixed at 4 hpi, and stained for Orf65+ viral particles (red) and nuclei (blue). (B, F and J) The total number of nuclei bearing at least one Orf65+ viral particle was determined and calculated as Orf65+ nuclei (%). In parallel experiments, cells were subjected to the same treatments and evaluated for viability by PI staining. (C, G and K) The total number of Orf65+ viral particles docked at each nucleus was determined. (D, H and L) Disruption of the actin cytoskeleton by cytochalasin (D), latrunculin A (H) and jasplakinolide (L). HUVEC were treated with the inhibitors as described in A, E and I, and stained for the actin cytoskeleton with AlexaFluor 488-phalloidin except for cells treated with jasplakinolide, which were stained with an antibody to β-actin. (M) Disruption of actin dynamics reduces the internalization of AlexaFluor 488-transferrin. HUVEC were pretreated with chemicals to disrupt actin dynamics for 1 h prior to exposure to AlexaFluor 488-transferrin. The reduced internalization and perinuclear accumulation of transferrin (green) compared to untreated control cells demonstrates the effective inhibition of clathrin-mediated endocytosis when actin dynamics are disrupted.

Article Snippet: AlexaFluor 488-phalloidin, AlexaFluor 488-transferrin, AlexaFluor 488-CTB, secondary antibodies AlexaFluor 568 goat-anti-mouse IgG1, AlexaFluor 568 and 647 goat anti-mouse IgG2a, and AlexaFluor 488 goat anti-rabbit IgG were from Molecular Probes, Invitrogen (Carlsbad, CA).

Techniques: Staining, Inhibition

Journal: PLoS Pathogens

Article Title: Actin Dynamics Regulate Multiple Endosomal Steps during Kaposi's Sarcoma-Associated Herpesvirus Entry and Trafficking in Endothelial Cells

doi: 10.1371/journal.ppat.1000512

Figure Lengend Snippet: Inhibition of clathrin assembly reduces the entry and trafficking of KSHV particles to the perinuclear region. (A and D) HUVEC were treated with chemical inhibitors of clathrin assembly at the indicated doses for 1 h prior to inoculation with KSHV. Cells were inoculated with KSHV in the presence of inhibitors, fixed at 8 hpi (dextrose) or 2 hpi (chlorpromazine), and stained for Orf65+ viral particles (red) and nuclei (blue). (B and E) The total number of nuclei bearing at least one Orf65+ particle was determined and calculated as Orf65+ nuclei (%). In parallel experiments, cells were subjected to the same treatments and evaluated for viability by PI staining. (C and F) The total number of Orf65+ particles docked at each nucleus was determined. (G–H) Inhibition of clathrin assembly reduces the internalization of AlexaFluor 488-transferrin (G) but not AlexaFluor 488-CTB (H). HUVEC were pretreated with inhibitors of clathrin assembly for 1 h prior to exposure to AlexaFluor 488-transferrin or AlexaFluor 488-CTB. The reduced internalization and perinuclear accumulation of transferrin (green) compared to untreated control cells demonstrates the effective inhibition of clathrin-mediated endocytosis while the internalization and perinuclear accumulation of CTB (green) indicates that the inhibitors did not affect caveolae-mediated endocytosis.

Article Snippet: AlexaFluor 488-phalloidin, AlexaFluor 488-transferrin, AlexaFluor 488-CTB, secondary antibodies AlexaFluor 568 goat-anti-mouse IgG1, AlexaFluor 568 and 647 goat anti-mouse IgG2a, and AlexaFluor 488 goat anti-rabbit IgG were from Molecular Probes, Invitrogen (Carlsbad, CA).

Techniques: Inhibition, Staining, CtB Assay

Journal: PLoS Pathogens

Article Title: Actin Dynamics Regulate Multiple Endosomal Steps during Kaposi's Sarcoma-Associated Herpesvirus Entry and Trafficking in Endothelial Cells

doi: 10.1371/journal.ppat.1000512

Figure Lengend Snippet: Colocalization of Orf65+ KSHV particles with markers of clathrin-mediated endocytosis during KSHV infection of endothelial cells. (A) HUVEC were inoculated with KSHV, fixed at 1 hpi and stained for clathrin heavy chain (red) and Orf65+ viral particles (green). (B) HUVEC were infected with KSHV and simultaneously labeled with Alexafluor 488-transferrin (green). Cells were fixed at 1 hpi and stained for Orf65+ viral particles (red). Regions highlighted in the squares in the upper images are shown as enlarged 3D-projection images in the lower panels. Areas of colocalization of red and green signals are revealed as yellow. For corresponding 3D projection of colocalization of Orf65+ particles with clathrin, see Videos S5 , S6 , S7 and S8 . For corresponding 3D projection of colocalization of Orf65+ particles with transferrin, see Videos S9 , S10 , S11 and S12 .

Article Snippet: AlexaFluor 488-phalloidin, AlexaFluor 488-transferrin, AlexaFluor 488-CTB, secondary antibodies AlexaFluor 568 goat-anti-mouse IgG1, AlexaFluor 568 and 647 goat anti-mouse IgG2a, and AlexaFluor 488 goat anti-rabbit IgG were from Molecular Probes, Invitrogen (Carlsbad, CA).

Techniques: Infection, Staining, Labeling

Journal: Beilstein Journal of Nanotechnology

Article Title: Nano-engineered skin mesenchymal stem cells: potential vehicles for tumour-targeted quantum-dot delivery

doi: 10.3762/bjnano.8.123

Figure Lengend Snippet: QD endocytic pathway in MSCs. QD uptake pathway in MSCs labelled with QDs in complete medium (a) or in serum-free medium (b). Uptake pathways were blocked using the endocytosis inhibitors CPZ, CytD, EIPA, nystatin and dynasore. Three overlaid channels represent Hoechst (blue), Phalloidin Alexa Fluor488 (green), carboxyl QD655 (yellow). Representative data are shown. QD fluorescence signal was quantified in complete (c) and in serum-free medium (d) cultivated MSCs. Statistical significance shown for the respective sample in comparison to control (Ctrl) sample; **p-value

Article Snippet: The cytoskeleton of cells was subsequently stained with methanolic Alexa Fluor488 Phalloidin (Thermo Fisher Scientific, USA) diluted 1:100 in washing buffer and incubated for 30 min at room temperature in the dark.

Techniques: Fluorescence

Journal: PLoS Pathogens

Article Title: Actin Dynamics Regulate Multiple Endosomal Steps during Kaposi's Sarcoma-Associated Herpesvirus Entry and Trafficking in Endothelial Cells

doi: 10.1371/journal.ppat.1000512

Figure Lengend Snippet: Disruption of actin regulators reduces the entry and trafficking of KSHV particles to the perinuclear region. (A and E) HUVEC were treated with chemicals to inhibit Rho GTPase function (A) or Arp2/3 complex activity (E) at the indicated doses for 1 h prior to inoculation with KSHV. Cells were inoculated with KSHV in the presence of inhibitors, fixed at 4 hpi, and stained for Orf65+ viral particles (red) and nuclei (blue). (B and F) The total number of nuclei bearing at least one Orf65+ particle was determined. In parallel experiments, cells were subjected to the same treatments and evaluated for viability by PI staining. (C and G) The total number of Orf65+ particles docked at each nucleus was determined. (D and H) Disruption of the actin cytoskeleton by CdTB (D) and wiskostatin (H). HUVEC were treated with the inhibitors as described in A and E, and stained for the actin cytoskeleton with AlexaFluor 488-phalloidin.

Article Snippet: AlexaFluor 488-phalloidin, AlexaFluor 488-transferrin, AlexaFluor 488-CTB, secondary antibodies AlexaFluor 568 goat-anti-mouse IgG1, AlexaFluor 568 and 647 goat anti-mouse IgG2a, and AlexaFluor 488 goat anti-rabbit IgG were from Molecular Probes, Invitrogen (Carlsbad, CA).

Techniques: Activity Assay, Staining

Journal: PLoS Pathogens

Article Title: Actin Dynamics Regulate Multiple Endosomal Steps during Kaposi's Sarcoma-Associated Herpesvirus Entry and Trafficking in Endothelial Cells

doi: 10.1371/journal.ppat.1000512

Figure Lengend Snippet: Disruption of actin dynamics reduces the entry and trafficking of KSHV particles to the perinuclear region. (A, E and I) HUVEC were treated with chemicals to disrupt actin dynamics at the indicated doses for 1 h. Cells were inoculated with KSHV in the presence of inhibitors, fixed at 4 hpi, and stained for Orf65+ viral particles (red) and nuclei (blue). (B, F and J) The total number of nuclei bearing at least one Orf65+ viral particle was determined and calculated as Orf65+ nuclei (%). In parallel experiments, cells were subjected to the same treatments and evaluated for viability by PI staining. (C, G and K) The total number of Orf65+ viral particles docked at each nucleus was determined. (D, H and L) Disruption of the actin cytoskeleton by cytochalasin (D), latrunculin A (H) and jasplakinolide (L). HUVEC were treated with the inhibitors as described in A, E and I, and stained for the actin cytoskeleton with AlexaFluor 488-phalloidin except for cells treated with jasplakinolide, which were stained with an antibody to β-actin. (M) Disruption of actin dynamics reduces the internalization of AlexaFluor 488-transferrin. HUVEC were pretreated with chemicals to disrupt actin dynamics for 1 h prior to exposure to AlexaFluor 488-transferrin. The reduced internalization and perinuclear accumulation of transferrin (green) compared to untreated control cells demonstrates the effective inhibition of clathrin-mediated endocytosis when actin dynamics are disrupted.

Article Snippet: AlexaFluor 488-phalloidin, AlexaFluor 488-transferrin, AlexaFluor 488-CTB, secondary antibodies AlexaFluor 568 goat-anti-mouse IgG1, AlexaFluor 568 and 647 goat anti-mouse IgG2a, and AlexaFluor 488 goat anti-rabbit IgG were from Molecular Probes, Invitrogen (Carlsbad, CA).

Techniques: Staining, Inhibition

Journal: Glycobiology

Article Title: Selective biochemical labeling of Campylobacter jejuni cell-surface glycoconjugates

doi: 10.1093/glycob/cwv016

Figure Lengend Snippet: Live-cell fluorescent labeling of C. jejuni . GalO was used to label wild-type and mutant strains with AlexaFluor 488 Hydroxylamine (488). Phase-contrast microscopy displays presence of cells (gray panels) while fluorescence microscopy indicates fluorescent labeling catalyzed by GalO (black panels).

Article Snippet: Cells were incubated with 30 µL catalase (Sigma-Aldrich, 4000 units/mL), 10 µL GalO (Worthington, 65 units/mL) and 0.5 µL alkoxyamine-PEG4-biotin (Pierce, 250 mM in DMSO) or 2.5 µL aminooxy-Flag (50 mM in water, synthesized according to ) or 2 µL AlexaFluor 488 Hydroxylamine (Life Technologies, 10 mM in H2 O) for 12 h at room temperature.

Techniques: Labeling, Mutagenesis, Microscopy, Fluorescence