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: 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: 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 Pathogens

Article Title: Lung macrophage scavenger receptor SR-A6 (MARCO) is an adenovirus type-specific virus entry receptor

doi: 10.1371/journal.ppat.1006914

Figure Lengend Snippet: SR-A6 facilitates virus binding to MPI-2 cells. A) Binding of Alexa-Fluor488-labeled HAdV-C5 to parental MPI-2 cells or the mSR-A6 -/- M3 and M2-4 cell lines. Virus was added to cells at +4°C for 60 min (moi ~ 2540 virus particles per cell) and cells were shifted to 37°C for 5min before analysis. The plot shows number of bound virus particles per cell, one dot representing one cell. Error bars represent the means ± SEMs. The difference between SR-A6-positive and SR-A6 knockout cells was statistically significant (P

Article Snippet: The viruses were isolated, and labeled with Alexa-Fluor488 (Thermo Fisher Scientific) or Atto565 (Sigma-Aldrich) as described [ – ].

Techniques: Binding Assay, Labeling, Knock-Out

Journal: PLoS Pathogens

Article Title: Lung macrophage scavenger receptor SR-A6 (MARCO) is an adenovirus type-specific virus entry receptor

doi: 10.1371/journal.ppat.1006914

Figure Lengend Snippet: High surface expression of human SR-A6 facilitates HAdV-C5 infection. A) Exogenous expression of human SR-A6 in L-929 cells (low CAR expression) promotes binding of HAdV-C5 to the cells. L-929 cells were transfected with a plasmid directing the synthesis of human SR-A6 from the cytomegalovirus major immediate early promoter and transfected cells were identified by immunostaining with anti-human SR-A6 antibody PLK1. Alexa-Fluor488-labeled HAdV-C5 particles were added to transfected L-929 cells at 4°C for 60 min. Fixed samples were imaged by confocal microscopy and virus particles associated with PLK1-positive and PLK1-negative cells were scored from maximum projections of confocal stacks. The plot shows number of virus particles per cell, one dot representing one cell. Horizontal bars represent mean values. Number of cells analyzed is indicated. The difference between PLK1-positive and -negative cells was statistically highly significant (P

Article Snippet: The viruses were isolated, and labeled with Alexa-Fluor488 (Thermo Fisher Scientific) or Atto565 (Sigma-Aldrich) as described [ – ].

Techniques: Expressing, Infection, Binding Assay, Transfection, Plasmid Preparation, Immunostaining, Labeling, Confocal Microscopy

Journal: PLoS Pathogens

Article Title: Lung macrophage scavenger receptor SR-A6 (MARCO) is an adenovirus type-specific virus entry receptor

doi: 10.1371/journal.ppat.1006914

Figure Lengend Snippet: Characterization of HAdV-C5 entry into MPI-2 cells. A) Internalization of HAdV-C5 into MPI-2 cells and B) exposure of viral membrane lytic protein VI on internalized virus. Atto565-labeled HAdV-C5 were added to MPI-2 cells at 4°C (moi ~13600 virus particles per cell) for 60 min. Unbound particles were washed away, and cells were shifted to 37°C for the indicated times. Intact cells were then incubated with 9C12 anti-hexon antibodies at 4°C to tag surface-associated viruses, and after fixation, cells were permeabilized and stained for protein VI. Secondary Alexa Fluor680-conjugated anti-mouse and Alexa-Fluor488-conjugated anti-rabbit antibodies were used to detect 9C12 and anti-protein VI antibodies, respectively. Nuclei were stained with DAPI, and samples were imaged by confocal microscopy. Virus particles lacking the 9C12 signal were scored as internalized particles and (A) shows percentage of internalized virus particles per cell at the different time points. One dot represents one cell. (B) shows mean average protein VI signal on internalized particles. One dot represents one cell. Error bars represent the means ± SEMs. Number of cells analyzed is indicated. C) SR-A6-facilitated entry supports efficient penetration of HAdV-C5 into the cytoplasm. Alexa-Fluor488-conjugated HAdV-C5 were added to MPI-2 cells at 4°C for 60 min (moi ~7300 virus particles per cell). Unbound viruses were washed away and cells were shifted to 37°C for 45 min. Cell surface and cytoplasmic particles were tagged with anti-Alexa-Fluor488 antibodies after perforation of the plasma membrane with streptolysin O (SLO). The anti-Alexa-Fluor488 antibodies in turn were visualized by secondary Alexa-Fluor594 antibodies. Control cells were incubated with antibodies without SLO treatment to specifically mark virus particles at the plasma membrane. The plot shows percentage of virus particles positive for the anti-Alexa-Fluor488 antibodies, one dot representing one cell. The majority of antibody-positive particles in the SLO-treated HAdV-C wild type (wt) sample represent cytoplasmic virus, since the no-SLO control indicated only few particles at the cell surface. Virus particles in the endosomes are inaccessible to the antibodies, and the endosomes stayed intact in the assay, since parallel samples infected with the penetration deficient HAdV-C2-TS1 mutant virus displayed only low number of antibody-positive particles. Error bars represent the means ± SEMs. Numbers of cells and virus particles analyzed are indicated. D) Tracking of incoming virus genome. EdC-labeled HAdV-C5 particles (moi ~ 7300 virus particles per cell) were internalized into MPI-2 cells at 37°C for 30 min and, after removal of unbound virus, the samples were incubated for further 270 min before fixation. The virus capsids were visualized by anti-hexon 9C12 and Alexa-Fluor594-conjugated secondary antibodies, and click-reaction with Alexa-Fluor488-conjugated azide was carried out to mark the virus genomes. The image represents maximum projection of image stack from central parts of the cells. Nuclear area and cell outline are indicated. In the majority of cells, the virus genome was separated from the capsid at this time point and concentrated over the nuclear area, as exemplified by the upper cell in the image. A fraction of cells displayed significant amounts of capsid-free virus DNA also in the cytoplasm, as exemplified by the lower cell in the image. Scale bar = 5 μm.

Article Snippet: The viruses were isolated, and labeled with Alexa-Fluor488 (Thermo Fisher Scientific) or Atto565 (Sigma-Aldrich) as described [ – ].

Techniques: Labeling, Incubation, Staining, Confocal Microscopy, Infection, Mutagenesis

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: 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