goat anti mouse alexa fluor 568 secondary antibody  (Thermo Fisher)


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    F ab 2 Goat anti Mouse IgG H L Cross Adsorbed Secondary Antibody
    Description:
    F ab 2 Goat anti Mouse IgG H L Cross Adsorbed Secondary Antibody for Western Blot IF ICC IHC Flow IP
    Catalog Number:
    31185
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    Category:
    Antibodies Secondary Detection Reagents
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    Structured Review

    Thermo Fisher goat anti mouse alexa fluor 568 secondary antibody
    Mesothelin is expressed in mesothelioma (MM) tumors. Paraffin sections of MM tumors derived from four different MM cell lines (PPM Mill, PPM Gat, PPM Gar, and HMESO) grown in severe combined immunodeficient (SCID) mice were stained with mouse anti-mesothelin antibody (clone MB) (A). <t>Alexa</t> <t>Fluor</t> 568 secondary antibody was used to visualize mesothelin (red), and SYTOX Green nucleic acid stain was used to visualize cell nuclei (green). Micrographs were taken at 400× magnification, scale bar = 50 µm. Mesothelin protein was assessed by Western blot analysis on human MM cell lines (B) and in human MM tumors grown either intraperitoneally (IP) or subcutaneously (SQ) (C). β-Actin was used as a loading control. Micrographs of Hema 3 differential stained cytospins from peritoneal lavage fluid (PLF) show uptake of acid-prepared mesoporous spheres (APMS)–MB and APMS-BSA microparticles ( n =5 mice/group). Particles surround the cell nuclei at 24 hr and 6 days (black arrows) (D). Micrographs were taken at 400× magnification, scale bar = 50 µm.
    F ab 2 Goat anti Mouse IgG H L Cross Adsorbed Secondary Antibody for Western Blot IF ICC IHC Flow IP
    https://www.bioz.com/result/goat anti mouse alexa fluor 568 secondary antibody/product/Thermo Fisher
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    goat anti mouse alexa fluor 568 secondary antibody - by Bioz Stars, 2021-06
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    Images

    1) Product Images from "A Multifunctional Mesothelin Antibody-tagged Microparticle Targets Human Mesotheliomas"

    Article Title: A Multifunctional Mesothelin Antibody-tagged Microparticle Targets Human Mesotheliomas

    Journal: Journal of Histochemistry and Cytochemistry

    doi: 10.1369/0022155412452567

    Mesothelin is expressed in mesothelioma (MM) tumors. Paraffin sections of MM tumors derived from four different MM cell lines (PPM Mill, PPM Gat, PPM Gar, and HMESO) grown in severe combined immunodeficient (SCID) mice were stained with mouse anti-mesothelin antibody (clone MB) (A). Alexa Fluor 568 secondary antibody was used to visualize mesothelin (red), and SYTOX Green nucleic acid stain was used to visualize cell nuclei (green). Micrographs were taken at 400× magnification, scale bar = 50 µm. Mesothelin protein was assessed by Western blot analysis on human MM cell lines (B) and in human MM tumors grown either intraperitoneally (IP) or subcutaneously (SQ) (C). β-Actin was used as a loading control. Micrographs of Hema 3 differential stained cytospins from peritoneal lavage fluid (PLF) show uptake of acid-prepared mesoporous spheres (APMS)–MB and APMS-BSA microparticles ( n =5 mice/group). Particles surround the cell nuclei at 24 hr and 6 days (black arrows) (D). Micrographs were taken at 400× magnification, scale bar = 50 µm.
    Figure Legend Snippet: Mesothelin is expressed in mesothelioma (MM) tumors. Paraffin sections of MM tumors derived from four different MM cell lines (PPM Mill, PPM Gat, PPM Gar, and HMESO) grown in severe combined immunodeficient (SCID) mice were stained with mouse anti-mesothelin antibody (clone MB) (A). Alexa Fluor 568 secondary antibody was used to visualize mesothelin (red), and SYTOX Green nucleic acid stain was used to visualize cell nuclei (green). Micrographs were taken at 400× magnification, scale bar = 50 µm. Mesothelin protein was assessed by Western blot analysis on human MM cell lines (B) and in human MM tumors grown either intraperitoneally (IP) or subcutaneously (SQ) (C). β-Actin was used as a loading control. Micrographs of Hema 3 differential stained cytospins from peritoneal lavage fluid (PLF) show uptake of acid-prepared mesoporous spheres (APMS)–MB and APMS-BSA microparticles ( n =5 mice/group). Particles surround the cell nuclei at 24 hr and 6 days (black arrows) (D). Micrographs were taken at 400× magnification, scale bar = 50 µm.

    Techniques Used: Derivative Assay, Mouse Assay, Staining, Western Blot

    2) Product Images from "Kinetic Evidence for Unique Regulation of GLUT4 Trafficking by Insulin and AMP-activated Protein Kinase Activators in L6 Myotubes *"

    Article Title: Kinetic Evidence for Unique Regulation of GLUT4 Trafficking by Insulin and AMP-activated Protein Kinase Activators in L6 Myotubes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.051185

    Transition to a steady-state GLUT4 distribution in L6 myotubes. A , L6 myotubes were stimulated with insulin (●), AICAR (■), A-769662 (▴), and insulin + AICAR (○) for the indicated times, and then cells were fixed, and the surface GLUT4 level was determined in fixed cells using an HA primary antibody and an Alexa 488 secondary antibody. Each data point represents the mean ± S.E. from three experiments. In some cases, the error bars are smaller than the symbol. B , times for the half-maximal increase above basal in cell surface HA-GLUT4 were determined for insulin, AICAR, A-769662, and the combination of insulin and AICAR. C , cell surface levels of GLUT4 reached at 30 min of incubation with the agonists were determined as a percentage of the total cellular GLUT4 available in permeabilized cells. D , plateau levels of antibody labeled-GLUT4 reached at 180 min of incubation with the anti-HA antibody were determined as a percentage of the total cellular GLUT4 available in permeabilized cells. Results are the mean ± S.E. from three experiments.
    Figure Legend Snippet: Transition to a steady-state GLUT4 distribution in L6 myotubes. A , L6 myotubes were stimulated with insulin (●), AICAR (■), A-769662 (▴), and insulin + AICAR (○) for the indicated times, and then cells were fixed, and the surface GLUT4 level was determined in fixed cells using an HA primary antibody and an Alexa 488 secondary antibody. Each data point represents the mean ± S.E. from three experiments. In some cases, the error bars are smaller than the symbol. B , times for the half-maximal increase above basal in cell surface HA-GLUT4 were determined for insulin, AICAR, A-769662, and the combination of insulin and AICAR. C , cell surface levels of GLUT4 reached at 30 min of incubation with the agonists were determined as a percentage of the total cellular GLUT4 available in permeabilized cells. D , plateau levels of antibody labeled-GLUT4 reached at 180 min of incubation with the anti-HA antibody were determined as a percentage of the total cellular GLUT4 available in permeabilized cells. Results are the mean ± S.E. from three experiments.

    Techniques Used: Incubation, Labeling

    HA antibody uptake reveals GLUT4 steady-state recycling kinetics. L6 myotubes were maintained in the basal state or stimulated to a steady state. Anti-HA antibody was then added, and the incubations with antibody were continued for the indicated times. At the indicated times, the bound antibody was determined by incubation with an Alexa 488 secondary antibody. A , L6 myotubes were maintained in the basal state or stimulated to steady state with insulin. At indicated times, L6 myotubes were visualized by wide field epifluorescence microscopy at ×40 magnification ( scale bar , 20 μm). B , L6 myotubes were maintained in the basal state (♦) or stimulated to steady state with insulin (●), AICAR (■), or insulin plus AICAR (○). Results are the mean ± S.E. from 3 to 7 experiments. In some cases error bars are smaller than the symbol.
    Figure Legend Snippet: HA antibody uptake reveals GLUT4 steady-state recycling kinetics. L6 myotubes were maintained in the basal state or stimulated to a steady state. Anti-HA antibody was then added, and the incubations with antibody were continued for the indicated times. At the indicated times, the bound antibody was determined by incubation with an Alexa 488 secondary antibody. A , L6 myotubes were maintained in the basal state or stimulated to steady state with insulin. At indicated times, L6 myotubes were visualized by wide field epifluorescence microscopy at ×40 magnification ( scale bar , 20 μm). B , L6 myotubes were maintained in the basal state (♦) or stimulated to steady state with insulin (●), AICAR (■), or insulin plus AICAR (○). Results are the mean ± S.E. from 3 to 7 experiments. In some cases error bars are smaller than the symbol.

    Techniques Used: Incubation, Epifluorescence Microscopy

    Akt control of the transition from basal to insulin-stimulated state. A , Akt activation was determined by blotting for phosphoserine 473 in basal cells and cells treated with insulin ( upper panels ). Both basal and insulin-treated cells were also treated with wortmannin ( WM ) and Akti as indicated. Akt2 levels were determined from the same solubilized cell lysates ( bottom panels ). B , rate of transition from the basal to the insulin-stimulated state in the absence (●) and presence of wortmannin (■), and Akti (▴) was determined in fixed cells using HA primary antibody and an Alexa 488 secondary antibody. Each point represents the mean ± S.E. from three experiments. In some cases error bars are smaller than the symbol.
    Figure Legend Snippet: Akt control of the transition from basal to insulin-stimulated state. A , Akt activation was determined by blotting for phosphoserine 473 in basal cells and cells treated with insulin ( upper panels ). Both basal and insulin-treated cells were also treated with wortmannin ( WM ) and Akti as indicated. Akt2 levels were determined from the same solubilized cell lysates ( bottom panels ). B , rate of transition from the basal to the insulin-stimulated state in the absence (●) and presence of wortmannin (■), and Akti (▴) was determined in fixed cells using HA primary antibody and an Alexa 488 secondary antibody. Each point represents the mean ± S.E. from three experiments. In some cases error bars are smaller than the symbol.

    Techniques Used: Activation Assay

    Direct measurements of steady-state GLUT4 internalization. A , L6 myotubes were maintained either in the basal steady state (♦) or in a steady state of stimulation with insulin (●), AICAR (■), or insulin plus AICAR (○). Anti-HA antibody was bound to cell surface GLUT4 at 4 °C; excess antibody was removed by washing, and then the cells were incubated for the indicated times at 37 °C. Surface-bound antibody was then removed, and the remaining antibody was detected with Alexa 488 secondary antibody. Results are the mean ± S.E. from 3 to 5 experiments. In some cases, error bars are smaller than the symbols. B , internalization k in rate constants from steady-state antibody uptake ( dark bars ) have been compared with the internalization assay measurements ( light bars ). Standard errors calculated from the confidence intervals (see “Experimental Procedures”) for the fits are shown.
    Figure Legend Snippet: Direct measurements of steady-state GLUT4 internalization. A , L6 myotubes were maintained either in the basal steady state (♦) or in a steady state of stimulation with insulin (●), AICAR (■), or insulin plus AICAR (○). Anti-HA antibody was bound to cell surface GLUT4 at 4 °C; excess antibody was removed by washing, and then the cells were incubated for the indicated times at 37 °C. Surface-bound antibody was then removed, and the remaining antibody was detected with Alexa 488 secondary antibody. Results are the mean ± S.E. from 3 to 5 experiments. In some cases, error bars are smaller than the symbols. B , internalization k in rate constants from steady-state antibody uptake ( dark bars ) have been compared with the internalization assay measurements ( light bars ). Standard errors calculated from the confidence intervals (see “Experimental Procedures”) for the fits are shown.

    Techniques Used: Incubation

    3) Product Images from "Novel Yttria-Stabilized Zirconium Oxide and Lithium Disilicate Coatings on Titanium Alloy Substrate for Implant Abutments and Biomedical Application"

    Article Title: Novel Yttria-Stabilized Zirconium Oxide and Lithium Disilicate Coatings on Titanium Alloy Substrate for Implant Abutments and Biomedical Application

    Journal: Materials

    doi: 10.3390/ma13092070

    Confocal laser scanning microscope (CLSM) images of immunohistochemically stained cells on surface coatings at a different time (2 and 24 h). HGF-1 nucleus (DAPI (4’,6-diamidino-2-phenylindole), blue), F-actin filaments (tetramethyl rhodamine iso-thiocyanate (TRITC)-conjugated phalloidin, red), and focal adhesion (FA) spots (vinculin stained with Alexa Fluor 488-conjugated antibodies, green).
    Figure Legend Snippet: Confocal laser scanning microscope (CLSM) images of immunohistochemically stained cells on surface coatings at a different time (2 and 24 h). HGF-1 nucleus (DAPI (4’,6-diamidino-2-phenylindole), blue), F-actin filaments (tetramethyl rhodamine iso-thiocyanate (TRITC)-conjugated phalloidin, red), and focal adhesion (FA) spots (vinculin stained with Alexa Fluor 488-conjugated antibodies, green).

    Techniques Used: Laser-Scanning Microscopy, Confocal Laser Scanning Microscopy, Staining

    4) Product Images from "Control of Spike Transfer at Hippocampal Mossy Fiber Synapses In Vivo by GABAA and GABAB Receptor-Mediated Inhibition"

    Article Title: Control of Spike Transfer at Hippocampal Mossy Fiber Synapses In Vivo by GABAA and GABAB Receptor-Mediated Inhibition

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2057-16.2016

    Electrophysiological characterization of GCs and CA3 PCs in vivo . A , Confocal fluorescent image of a DG cell filled with biocytin during whole-cell recording and visualized by post hoc labeling with Alexa Fluor 488. Scale bar, 10 μm. Dendritic spines from the same cells are plotted at higher magnification. Inset scale bar, 20 μm. The bottom part shows a mossy fiber bouton from the same DG cell in stratum lucidum. Scale bar, 5 μm. B , Example trace of a current-clamp recording from a DG cell showing lack of spontaneous firing, membrane potential oscillations (top), and subthreshold and suprathreshold responses to injected current pulses (bottom). C , Confocal fluorescent image of a CA3 PC filled with biocytin during whole-cell recording and visualized by post hoc labeling with Alexa Fluor 488. Scale bar, 10 μm. D , Example trace of a current-clamp recording from a CA3 PC showing spontaneous single and bursts of action potentials (arrow). E , Example of burst of action potentials obtained from traces shown in D . F , Pie chart showing the proportion of single action potentials vs bursts in CA3 PCs. G , Scatter plot graph summarizing resting membrane potential (left) and ( H ) input resistance (right) of GCs ( n = 12) and CA3 PCs ( n = 20).
    Figure Legend Snippet: Electrophysiological characterization of GCs and CA3 PCs in vivo . A , Confocal fluorescent image of a DG cell filled with biocytin during whole-cell recording and visualized by post hoc labeling with Alexa Fluor 488. Scale bar, 10 μm. Dendritic spines from the same cells are plotted at higher magnification. Inset scale bar, 20 μm. The bottom part shows a mossy fiber bouton from the same DG cell in stratum lucidum. Scale bar, 5 μm. B , Example trace of a current-clamp recording from a DG cell showing lack of spontaneous firing, membrane potential oscillations (top), and subthreshold and suprathreshold responses to injected current pulses (bottom). C , Confocal fluorescent image of a CA3 PC filled with biocytin during whole-cell recording and visualized by post hoc labeling with Alexa Fluor 488. Scale bar, 10 μm. D , Example trace of a current-clamp recording from a CA3 PC showing spontaneous single and bursts of action potentials (arrow). E , Example of burst of action potentials obtained from traces shown in D . F , Pie chart showing the proportion of single action potentials vs bursts in CA3 PCs. G , Scatter plot graph summarizing resting membrane potential (left) and ( H ) input resistance (right) of GCs ( n = 12) and CA3 PCs ( n = 20).

    Techniques Used: In Vivo, Labeling, Injection

    5) Product Images from "Creation of Dystrophin Expressing Chimeric Cells of Myoblast Origin as a Novel Stem Cell Based Therapy for Duchenne Muscular Dystrophy"

    Article Title: Creation of Dystrophin Expressing Chimeric Cells of Myoblast Origin as a Novel Stem Cell Based Therapy for Duchenne Muscular Dystrophy

    Journal: Stem Cell Reviews

    doi: 10.1007/s12015-017-9792-7

    In vitro analysis of proliferation kinetics and myogenic differentiation of murine Dystrophin Expressing Chimeric Cells (DEC). a  The DEC proliferation kinetics after fusion compared with  snj  wild type myoblasts (MB wt ) and dystrophin deficient  mdx  (MB mdx ) parent cells proliferation up to 21 days of culturing. Absolute cell counts (n = 3/cell type) at each time point (day 0, 3, 6, 9, 12, 15, 18, 21) were normalized with number of seeded cell and proliferation was expressed in fold increase. MB wt /MB mdx  DEC cell proliferation results did not show differences in the maximal proliferation counts after fusion when compared to parent myoblast populations (p  >  0.05, one-way ANOVA, not significant), confirming maintenance of efficient cell cycle with maximal proliferation peak reached 3 days earlier compared to not-fused controls.  b  Representative immunofluorescence images of DEC differentiated into skeletal myocytes expressing skeletal myosin heavy chain marker (SMHC-AlexaFluor 647, yellow) seven days after fusion. Confirmation of differentiation of MB wt /MB mdx  DEC line to the skeletal myocytes in myogenic differentiation media comparable with MB wt  controls. For merge: Yellow, SMHC; blue, DAPI (nuclei), scale bar 10 μm
    Figure Legend Snippet: In vitro analysis of proliferation kinetics and myogenic differentiation of murine Dystrophin Expressing Chimeric Cells (DEC). a The DEC proliferation kinetics after fusion compared with snj wild type myoblasts (MB wt ) and dystrophin deficient mdx (MB mdx ) parent cells proliferation up to 21 days of culturing. Absolute cell counts (n = 3/cell type) at each time point (day 0, 3, 6, 9, 12, 15, 18, 21) were normalized with number of seeded cell and proliferation was expressed in fold increase. MB wt /MB mdx DEC cell proliferation results did not show differences in the maximal proliferation counts after fusion when compared to parent myoblast populations (p  >  0.05, one-way ANOVA, not significant), confirming maintenance of efficient cell cycle with maximal proliferation peak reached 3 days earlier compared to not-fused controls. b Representative immunofluorescence images of DEC differentiated into skeletal myocytes expressing skeletal myosin heavy chain marker (SMHC-AlexaFluor 647, yellow) seven days after fusion. Confirmation of differentiation of MB wt /MB mdx DEC line to the skeletal myocytes in myogenic differentiation media comparable with MB wt controls. For merge: Yellow, SMHC; blue, DAPI (nuclei), scale bar 10 μm

    Techniques Used: In Vitro, Expressing, Immunofluorescence, Marker

    6) Product Images from "DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur"

    Article Title: DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200609014

    Notch1 transendocytosis and signaling require general endocytic machinery. (A) Dll1 cells were transiently transfected with EGFP, dynaminK44A-EGFP, or EGFP-Eps15DIII, and then cocultured with HA-N1 cells. Cocultures were fixed, permeabilized, and stained with a mouse HA antibody (262K) and anti–mouse conjugated to Alexa Fluor 568 to detect the N1 N terminus (red), followed by rabbit anti-GFP conjugated to Alexa Fluor 488 to detect EGFP (green). Arrows indicate interacting N1 N terminus. Arrowheads indicate N1 N terminus associated with Dll1 cells. An untransfected Dll1 cell is outlined. Fluorescent images are confocal projections through the midsection of the Dll1 cell. (B) Transfer of N1 N terminus was quantified by examining Dll1 cells displaying EGFP fluorescence for internal N1 N terminus (see Materials and methods). (C) HA-N1 cells transfected with a CSL-luciferase reporter were cocultured with HEK 293-T cells cotransfected with Dll1 and EGFP, dynaminK44A-EGFP, or EGFP-Eps15DIII and assayed for luciferase activity. Values are fold-induction over vector + EGFP-transfected cells. (D) HA-N1 cells were cocultured with Dll1 cells in the presence of transferrin conjugated to FITC (green). Cocultures were fixed, permeabilized, and stained with a mouse HA antibody (262K) and anti–mouse conjugated to Alexa Fluor 568 to detect the N1 N terminus (red). Arrowheads indicate colocalization of transferrin and N1 N terminus in Dll1 cells (yellow). Boxes denote enlarged region. Low magnification fluorescent images are confocal projections, and enlargements are a 0.34 μm confocal section through the midsection of the Dll1 cell. Error bars represent the SEM (B) and the SD (C). *, P
    Figure Legend Snippet: Notch1 transendocytosis and signaling require general endocytic machinery. (A) Dll1 cells were transiently transfected with EGFP, dynaminK44A-EGFP, or EGFP-Eps15DIII, and then cocultured with HA-N1 cells. Cocultures were fixed, permeabilized, and stained with a mouse HA antibody (262K) and anti–mouse conjugated to Alexa Fluor 568 to detect the N1 N terminus (red), followed by rabbit anti-GFP conjugated to Alexa Fluor 488 to detect EGFP (green). Arrows indicate interacting N1 N terminus. Arrowheads indicate N1 N terminus associated with Dll1 cells. An untransfected Dll1 cell is outlined. Fluorescent images are confocal projections through the midsection of the Dll1 cell. (B) Transfer of N1 N terminus was quantified by examining Dll1 cells displaying EGFP fluorescence for internal N1 N terminus (see Materials and methods). (C) HA-N1 cells transfected with a CSL-luciferase reporter were cocultured with HEK 293-T cells cotransfected with Dll1 and EGFP, dynaminK44A-EGFP, or EGFP-Eps15DIII and assayed for luciferase activity. Values are fold-induction over vector + EGFP-transfected cells. (D) HA-N1 cells were cocultured with Dll1 cells in the presence of transferrin conjugated to FITC (green). Cocultures were fixed, permeabilized, and stained with a mouse HA antibody (262K) and anti–mouse conjugated to Alexa Fluor 568 to detect the N1 N terminus (red). Arrowheads indicate colocalization of transferrin and N1 N terminus in Dll1 cells (yellow). Boxes denote enlarged region. Low magnification fluorescent images are confocal projections, and enlargements are a 0.34 μm confocal section through the midsection of the Dll1 cell. Error bars represent the SEM (B) and the SD (C). *, P

    Techniques Used: Transfection, Staining, Fluorescence, Luciferase, Activity Assay, Plasmid Preparation

    Transendocytosed Notch1 structures are internal and disconnected from the plasma membrane. (A) Staining protocol to distinguish surface and internal N1 (see Materials and methods for details). (B) Validation of protocol in A. L cells expressing Dll1 with HA tags on either the intracellular (Dll1-HA) or extracellular (HA-Dll1) domain were fixed and stained for surface HA with mouse HA antibody (262K) and anti–mouse Alexa Fluor 568 (red). After permeabilization, cells were stained for total HA (surface and intracellular) with an HA antibody (16B12) conjugated to Alexa Fluor 488 (green) and imaged by confocal and DIC microscopy. (C) Cocultures of HA-N1 cells with Dll1 or J1 cells were fixed and stained with rabbit anti-ECD antibodies to Dll1 or J1 and anti–rabbit Alexa Fluor 633 antibodies (blue) to label the surface of the ligand cell, followed by staining for surface N1 N terminus with a mouse HA antibody (262K) and anti–mouse Alexa Fluor 568 (red). After permeabilization, cells were stained for total N1 N terminus (surface and intracellular) with an HA antibody (16B12) conjugated to Alexa Fluor 488 (green), and imaged by confocal and DIC microscopy. Arrows indicate N1 N terminus on both the Notch cell surface (yellow) and the ligand cell surface (white). Arrowheads indicate internal N1 N terminus detected within ligand cells (green). Boxes denote enlarged regions. (D) Transendocytosis was quantified by examining Dll1 or J1 cells for N1 N terminus detected exclusively after permeabilization (see Materials and methods). Error bars represent the SEM. Images from each experiment were uniformly adjusted using the levels function in Photoshop. Bars, 5 μm.
    Figure Legend Snippet: Transendocytosed Notch1 structures are internal and disconnected from the plasma membrane. (A) Staining protocol to distinguish surface and internal N1 (see Materials and methods for details). (B) Validation of protocol in A. L cells expressing Dll1 with HA tags on either the intracellular (Dll1-HA) or extracellular (HA-Dll1) domain were fixed and stained for surface HA with mouse HA antibody (262K) and anti–mouse Alexa Fluor 568 (red). After permeabilization, cells were stained for total HA (surface and intracellular) with an HA antibody (16B12) conjugated to Alexa Fluor 488 (green) and imaged by confocal and DIC microscopy. (C) Cocultures of HA-N1 cells with Dll1 or J1 cells were fixed and stained with rabbit anti-ECD antibodies to Dll1 or J1 and anti–rabbit Alexa Fluor 633 antibodies (blue) to label the surface of the ligand cell, followed by staining for surface N1 N terminus with a mouse HA antibody (262K) and anti–mouse Alexa Fluor 568 (red). After permeabilization, cells were stained for total N1 N terminus (surface and intracellular) with an HA antibody (16B12) conjugated to Alexa Fluor 488 (green), and imaged by confocal and DIC microscopy. Arrows indicate N1 N terminus on both the Notch cell surface (yellow) and the ligand cell surface (white). Arrowheads indicate internal N1 N terminus detected within ligand cells (green). Boxes denote enlarged regions. (D) Transendocytosis was quantified by examining Dll1 or J1 cells for N1 N terminus detected exclusively after permeabilization (see Materials and methods). Error bars represent the SEM. Images from each experiment were uniformly adjusted using the levels function in Photoshop. Bars, 5 μm.

    Techniques Used: Staining, Expressing, Microscopy

    Notch1 transendocytosis, hNotch1 subunit separation, and signaling require Dll1 endocytosis. (A) Dll1, Dll1ΔICD, or parental L cells were incubated with rabbit Dll1 extracellular domain (ECD) antibodies and N1Fc to track Dll1. After incubation, the cells were fixed, permeabilized, and stained with anti–rabbit Alexa Fluor 488 to detect Dll1 antibodies (green) and anti–human Fc conjugated to Cy5 (red) to detect N1Fc. (B) Dll1 or Dll1ΔICD cells were incubated with N1Fc preclustered with anti–human Fc conjugated to Texas red (red), and transferrin conjugated to FITC (green). Arrowhead indicates internalized N1Fc colocalized with transferrin in Dll1 cells (yellow). Arrow indicates N1Fc on the surface of Dll1ΔICD cells (red). Asterisk indicates internalized transferrin. (C) Cocultures of HA-N1 cells and Dll1ΔICD or Dll10CD cells were treated as in Fig. 2 C to identify surface mutant Dll1 (blue), surface N1 N terminus (red), and total N1 N terminus (green). Arrows indicate N1 N terminus on the surface of the Notch cell (yellow), as well as the mutant ligand cell (white). (D) Transendocytosis was quantified as in Fig. 2 D . (E) Cocultures of HA-N1-EGFP and Dll1ΔICD cells were treated as in Fig. 4 A to identify N1 N terminus (red) and N1 C terminus (green). Arrow indicates double-positive HA-N1-EGFP cluster (yellow). (F) Dissociation ratio was quantified as in Fig. 4 B . (G) Dll1ΔICD cells were cocultured with HA-N1 cells and treated as in Fig. 3 A to identify N1 N terminus (green) and activated NICD (red). (H) NICD-positive nuclei were scored as in Fig. 3 B . (I) CSL reporter assay, as in Fig. 3 C . Cells in A–C, E, and G were imaged by confocal and DIC microscopy. Boxes indicate enlarged regions. Overlays are composites of fluorescent and DIC images. *, P
    Figure Legend Snippet: Notch1 transendocytosis, hNotch1 subunit separation, and signaling require Dll1 endocytosis. (A) Dll1, Dll1ΔICD, or parental L cells were incubated with rabbit Dll1 extracellular domain (ECD) antibodies and N1Fc to track Dll1. After incubation, the cells were fixed, permeabilized, and stained with anti–rabbit Alexa Fluor 488 to detect Dll1 antibodies (green) and anti–human Fc conjugated to Cy5 (red) to detect N1Fc. (B) Dll1 or Dll1ΔICD cells were incubated with N1Fc preclustered with anti–human Fc conjugated to Texas red (red), and transferrin conjugated to FITC (green). Arrowhead indicates internalized N1Fc colocalized with transferrin in Dll1 cells (yellow). Arrow indicates N1Fc on the surface of Dll1ΔICD cells (red). Asterisk indicates internalized transferrin. (C) Cocultures of HA-N1 cells and Dll1ΔICD or Dll10CD cells were treated as in Fig. 2 C to identify surface mutant Dll1 (blue), surface N1 N terminus (red), and total N1 N terminus (green). Arrows indicate N1 N terminus on the surface of the Notch cell (yellow), as well as the mutant ligand cell (white). (D) Transendocytosis was quantified as in Fig. 2 D . (E) Cocultures of HA-N1-EGFP and Dll1ΔICD cells were treated as in Fig. 4 A to identify N1 N terminus (red) and N1 C terminus (green). Arrow indicates double-positive HA-N1-EGFP cluster (yellow). (F) Dissociation ratio was quantified as in Fig. 4 B . (G) Dll1ΔICD cells were cocultured with HA-N1 cells and treated as in Fig. 3 A to identify N1 N terminus (green) and activated NICD (red). (H) NICD-positive nuclei were scored as in Fig. 3 B . (I) CSL reporter assay, as in Fig. 3 C . Cells in A–C, E, and G were imaged by confocal and DIC microscopy. Boxes indicate enlarged regions. Overlays are composites of fluorescent and DIC images. *, P

    Techniques Used: Incubation, Staining, Mutagenesis, Reporter Assay, Microscopy

    DSL ligands mediate clustering and transendocytosis of Notch1 to activate Notch1 proteolysis and downstream signaling. (A) HA-N1 cells cocultured with Dll1, J1, or parental L cells were fixed, permeabilized, and stained with an HA antibody (16B12) conjugated to Alexa Fluor 488 to detect the N1 N terminus (green), followed by rabbit antibodies to activated NICD (Val1744) and anti–rabbit Alexa Fluor 568 (red) and imaged by confocal and DIC microscopy. Arrows indicate N1 N terminus polarized to sites of contact between ligand and N1 cells. Arrowheads indicate N1 N terminus within ligand cells interacting with N1 cells and displaying a signal for activated NICD in the nucleus. Boxes denote enlarged regions; overlays are composites of fluorescent and DIC images. A low level of red-channel nonnuclear background fluorescence that is insensitive to DAPT ( Fig. 5 E ) is detected with all L-cell lines. (B) Cells displaying transfer of N-terminal puncta to Dll1 or J1 cells were scored for NICD-positive nuclei. (C) HA-N1 cells transfected with a CSL-luciferase reporter were cocultured with ligand cells and assayed for luciferase activity. Values represent fold-induction over cocultures with parental L cells. RLU, relative luciferase units. Error bars represent the SEM (B) and the SD (C). Images were uniformly adjusted using the levels function in Photoshop. Bar, 5 μm.
    Figure Legend Snippet: DSL ligands mediate clustering and transendocytosis of Notch1 to activate Notch1 proteolysis and downstream signaling. (A) HA-N1 cells cocultured with Dll1, J1, or parental L cells were fixed, permeabilized, and stained with an HA antibody (16B12) conjugated to Alexa Fluor 488 to detect the N1 N terminus (green), followed by rabbit antibodies to activated NICD (Val1744) and anti–rabbit Alexa Fluor 568 (red) and imaged by confocal and DIC microscopy. Arrows indicate N1 N terminus polarized to sites of contact between ligand and N1 cells. Arrowheads indicate N1 N terminus within ligand cells interacting with N1 cells and displaying a signal for activated NICD in the nucleus. Boxes denote enlarged regions; overlays are composites of fluorescent and DIC images. A low level of red-channel nonnuclear background fluorescence that is insensitive to DAPT ( Fig. 5 E ) is detected with all L-cell lines. (B) Cells displaying transfer of N-terminal puncta to Dll1 or J1 cells were scored for NICD-positive nuclei. (C) HA-N1 cells transfected with a CSL-luciferase reporter were cocultured with ligand cells and assayed for luciferase activity. Values represent fold-induction over cocultures with parental L cells. RLU, relative luciferase units. Error bars represent the SEM (B) and the SD (C). Images were uniformly adjusted using the levels function in Photoshop. Bar, 5 μm.

    Techniques Used: Staining, Microscopy, Fluorescence, Transfection, Luciferase, Activity Assay

    DSL ligand–induced transendocytosis promotes separation of the Notch1 N- and C-terminal subunits. (A) HA-N1-EGFP C2C12 cells were cocultured with Dll1, J1, or parental L cells. Cocultures were fixed, permeabilized, and stained with a mouse HA antibody (262K) and anti–mouse Alexa Fluor 568 to detect the N1 N terminus (red) and a rabbit anti-GFP antibody conjugated to Alexa Fluor 488 to detect the N1 C terminus (green), and then imaged by confocal and DIC microscopy. Arrows indicate double-positive HA-N1-EGFP clusters at interfaces between N1 and ligand cells. Arrowheads indicate puncta positive only for the N1 N terminus within ligand cells. Boxes denote enlarged regions; overlays are composites of fluorescent and DIC images. (B) The signals for Notch N and C termini were divided to produce the dissociation ratio (see Materials and methods) for ligand and Notch cells. Error bars represent the SEM. *, P
    Figure Legend Snippet: DSL ligand–induced transendocytosis promotes separation of the Notch1 N- and C-terminal subunits. (A) HA-N1-EGFP C2C12 cells were cocultured with Dll1, J1, or parental L cells. Cocultures were fixed, permeabilized, and stained with a mouse HA antibody (262K) and anti–mouse Alexa Fluor 568 to detect the N1 N terminus (red) and a rabbit anti-GFP antibody conjugated to Alexa Fluor 488 to detect the N1 C terminus (green), and then imaged by confocal and DIC microscopy. Arrows indicate double-positive HA-N1-EGFP clusters at interfaces between N1 and ligand cells. Arrowheads indicate puncta positive only for the N1 N terminus within ligand cells. Boxes denote enlarged regions; overlays are composites of fluorescent and DIC images. (B) The signals for Notch N and C termini were divided to produce the dissociation ratio (see Materials and methods) for ligand and Notch cells. Error bars represent the SEM. *, P

    Techniques Used: Staining, Microscopy

    DSL ligands induce clustering and transendocytosis of Notch1. (A) Coculture and staining protocol for Dll1 endocytosis and N1 transendocytosis (see Materials and methods for details). Small filled circles represent intracellular vesicles. (B and C) HA-N1 C2C12 cells were cocultured with Dll1 (B) or parental L (C) cells in the presence of rabbit anti-Dll1 extracellular domain (ECD) antibodies to track Dll1 internalization. Cells were then fixed, permeabilized, stained with an HA antibody (16B12) conjugated to Alexa Fluor 488 to detect N1 N terminus (green) and anti–rabbit Alexa Fluor 633 to detect Dll1 antibodies (red), and imaged by confocal and differential interference contrast (DIC) microscopy. Arrows indicate N1 puncta polarized at interfaces of Dll1–N1 cells; arrowheads indicate colocalization of N1 and Dll1 within the Dll1 cell (yellow). Boxes indicate enlarged regions. Overlays are composites of fluorescent and DIC images. Images were uniformly adjusted using the levels function in Photoshop (Adobe). Bar, 5 μm.
    Figure Legend Snippet: DSL ligands induce clustering and transendocytosis of Notch1. (A) Coculture and staining protocol for Dll1 endocytosis and N1 transendocytosis (see Materials and methods for details). Small filled circles represent intracellular vesicles. (B and C) HA-N1 C2C12 cells were cocultured with Dll1 (B) or parental L (C) cells in the presence of rabbit anti-Dll1 extracellular domain (ECD) antibodies to track Dll1 internalization. Cells were then fixed, permeabilized, stained with an HA antibody (16B12) conjugated to Alexa Fluor 488 to detect N1 N terminus (green) and anti–rabbit Alexa Fluor 633 to detect Dll1 antibodies (red), and imaged by confocal and differential interference contrast (DIC) microscopy. Arrows indicate N1 puncta polarized at interfaces of Dll1–N1 cells; arrowheads indicate colocalization of N1 and Dll1 within the Dll1 cell (yellow). Boxes indicate enlarged regions. Overlays are composites of fluorescent and DIC images. Images were uniformly adjusted using the levels function in Photoshop (Adobe). Bar, 5 μm.

    Techniques Used: Staining, Microscopy

    7) Product Images from "Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls"

    Article Title: Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2018.07.004

    Immunofluorescence Analysis of Intracellular Localization of AAV8 Particles (A) Representative pictures of infected HeLa cells. Cells were non-infected (no AAV) or infected with AAV8 control or AAV8ΔVP1 at a multiplicity of 20,000 VG/cell, and then they were fixed after 1, 5, or 16 hr. Cell nuclei stained with DraQ5 appear in red, and assembled AAV8 particles stained with Alexa Fluor 555 appear in blue, green, or cyan, depending on their localization (cytoplasmic, intranuclear, or perinuclear, respectively). (B) Quantitative analysis of the immunofluorescence pictures. AAV8-assembled particles were quantified in the intranuclear, perinuclear, and cytoplasmic cellular compartments at 1, 5, and 16 hr post-infection with AAV8-GFP (upper panel) or AAV8ΔVP1-GFP (lower panel). Results obtained with AAV8ΔVP1 and AAV8 were compared by a two-tailed Mann-Whitney test for each cell compartment. *p
    Figure Legend Snippet: Immunofluorescence Analysis of Intracellular Localization of AAV8 Particles (A) Representative pictures of infected HeLa cells. Cells were non-infected (no AAV) or infected with AAV8 control or AAV8ΔVP1 at a multiplicity of 20,000 VG/cell, and then they were fixed after 1, 5, or 16 hr. Cell nuclei stained with DraQ5 appear in red, and assembled AAV8 particles stained with Alexa Fluor 555 appear in blue, green, or cyan, depending on their localization (cytoplasmic, intranuclear, or perinuclear, respectively). (B) Quantitative analysis of the immunofluorescence pictures. AAV8-assembled particles were quantified in the intranuclear, perinuclear, and cytoplasmic cellular compartments at 1, 5, and 16 hr post-infection with AAV8-GFP (upper panel) or AAV8ΔVP1-GFP (lower panel). Results obtained with AAV8ΔVP1 and AAV8 were compared by a two-tailed Mann-Whitney test for each cell compartment. *p

    Techniques Used: Immunofluorescence, Infection, Staining, Two Tailed Test, MANN-WHITNEY

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    Incubation:

    Article Title: Kinetic Evidence for Unique Regulation of GLUT4 Trafficking by Insulin and AMP-activated Protein Kinase Activators in L6 Myotubes *
    Article Snippet: Cells were subsequently fixed but not permeabilized, and the amount of HA-GLUT4 present at the plasma membrane was determined from the accessibility of the HA epitope to anti-HA antibody (Covance). .. Finally, cells were incubated with 20 μg/ml goat anti-mouse Alexa 488-conjugated secondary antibody (Molecular Probes, Invitrogen). .. After washing, fluorescence (emission 485 nm/excitation 520 nm) was measured in bottom reading mode using a fluorescent microtiter plate reader (FLUOstar Galaxy; BMG Labtechnologies).

    Article Title: Novel Yttria-Stabilized Zirconium Oxide and Lithium Disilicate Coatings on Titanium Alloy Substrate for Implant Abutments and Biomedical Application
    Article Snippet: Soon after, specimens were washed three times for 5 min with a 0.05% Tween-20 solution. .. They were then incubated with secondary goat anti-mouse Alexa Fluor 488-conjugated antibodies (Invitrogen, Carlsbad, CA, USA) and tetramethyl rhodamine iso-thiocyanate (TRITC)-conjugated phalloidin (1:500; Merck Millipore, Carlsbad, CA, USA) in PBS in a darkened environment at RT for 1 h with 25 rpm agitation. .. After that, the samples were washed three more times with PBS for 5 min at RT and stained with 12.5 µg/mL 4’,6-diamidino-2-phenylindole (DAPI; Merck Millipore, Carlsbad, CA, USA) solution in PBS for 5 min in the dark at RT with 25 rpm agitation.

    Article Title: Engagement of Nucleotide-binding Oligomerization Domain-containing Protein 1 (NOD1) by Receptor-interacting Protein 2 (RIP2) Is Insufficient for Signal Transduction *
    Article Snippet: After 24 h, cells were fixed with 3% paraformaldehyde (Roth) in PBS for 10 min and permeabilized with 0.5% Triton X-100 (Roth) in cold PBS for 5 min. .. Cells were blocked in 3% BSA (Roth) in PBS for 20 min and incubated successively in mouse anti-FLAG M2 (1:20,000; Stratagene) and goat anti-mouse Alexa Fluor 546 (1: 200; Invitrogen Molecular Probes) antibodies. .. DNA was stained with DAPI (5 μg/ml; Invitrogen Molecular Probes), and actin was stained with phalloidin-FITC (2.5 μg/ml; Sigma-Aldrich).

    Fluorescence:

    Article Title: Control of Spike Transfer at Hippocampal Mossy Fiber Synapses In Vivo by GABAA and GABAB Receptor-Mediated Inhibition
    Article Snippet: Slices were treated in a blocking buffer [5% normal goat serum in 0.3% PBS–Tween 20 (PBS-T)] for 45 min and incubated with primary antibody solution for 48 h. Slices were then washed and incubated for 2–3 h with a secondary antibody. .. EYFP fluorescence from ChR2+ GCs was enhanced using a mouse anti-GFP (1:500; catalog #11814460, Sigma-Aldrich) and goat anti-mouse conjugated to Alexa Fluor 488 (1:500; catalog # , Thermo Fisher Scientific) diluted in PBS-T. A mounting medium containing DAPI was used (VECTASHIELD, Vector Laboratories). .. The slide scanner was a Nanozoomer 2.0HT with a fluorescence imaging module (Hamamatsu Photonics) using a planar apochromatic 20 × objective with NA of 0.75 combined with an additional lens 1.75×, with a 2 × 2 binning leading to a final magnification of 20×.

    Labeling:

    Article Title: Revealing microbial recognition by specific antibodies
    Article Snippet: Flow cytometry Samples were suspended in sterile saline solution with 5 % albumin to prevent non-specific antibody binding, then stained with (i) anti-human IgA or IgG labelled with FITC (Invitrogen catalog # A18782 and A18806); and (ii) the DNA-binding fluorophor SYTO62 (Invitrogen catalog # S11344) according to the manufacturer instructions. .. Anti-mouse IgA or IgG labeled with FITC (Invitrogen catalog # M31101 and A24525) were used for isotype controls. .. Cell sorting was performed with the MoFloTM XDP flow cytometer (Beckman Coulter Inc.) using Argon 488 nm (blue) laser (200 mW power) and the 635 nm (red) diode laser (25 mW power) as light sources.

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    Thermo Fisher texas redx conjugated goat anti mouse igg
    Validation of external S. aureus labeling. Adherent GM-MФs were either treated with cytochalasin D (to block uptake) or control prior to and during incubation with GFP- S. aureus RN6390 and incubated with lysostaphin (to degrade external S. aureus , eliminating external bacteria) or control following incubation with S. aureus . External S. aureus were labeled with an <t>IgG</t> 3 monoclonal mouse anti- S. aureus primary antibody and <t>Texas-RedX-conjugated</t> goat anti-mouse secondary antibody. (A) No treatment. (B) Treatment with cytochalasin D. (C) Treatment with lysostaphin. (D) Treatment with both cytochalasin D and lysostaphin. (Original magnification, 200X).
    Texas Redx Conjugated Goat Anti Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa 488 conjugated secondary anti goat antibodies
    Expression analysis of Wnt/β-catenin target genes, CD44 and EphB2, in the gastrointestinal tract of Ad Dkk1- or Ad Fc-treated adult C57BL/6 mice (12–16 weeks old). Organs were harvested 2 days after Ad Dkk1 i.v injection (10 9 pfu). ( Left ) Ad Dkk1 repression of CD44 expression in proliferative zones of all levels of the gastrointestinal epithelium. Arrowheads indicate the absence of CD44 immunoreactivity in proliferative compartments of the intestinal epithelium in Ad Dkk1 animals. * , residual CD44 staining in nonepithelial lamina propria. ( Right ) Ad Dkk1 repression of EphB2 in small intestine and colon. Repression was weaker in ascending colon and no repression was observed in stomach. EphB2 immunofluorescence was performed with <t>Alexa</t> 488 detection of EphB2 immunoreactivity (green) and Hoechst 33342 nuclear counterstain (blue). Stomach (st), duodenum (du), jejunum (je), ileum (il), cecum (ce), ascending colon (ac), and descending colon (dc) are shown.
    Alexa 488 Conjugated Secondary Anti Goat Antibodies, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti mouse fitc
    US27 is found in close proximity to <t>CXCR4.</t> (A) Immunofluorescence microscopy of HEK293-US27 or HEK293-US28 cells stained with anti-FLAG (green) and anti-CXCR4 (red) followed by goat anti-mouse <t>FITC</t> and donkey anti-goat TRITC secondary antibodies, respectively. Nuclei were visualized with DAPI (blue). Bar, 10 μm. (B) HEK293-US27 or HEK293-US28 cells were stained with anti-FLAG and anti-CXCR4 followed by oligonucleotide-conjugated secondary antibodies and amplification using the DuoLink kit for the proximity ligation assay (PLA). Red PLA spots indicate receptors in close proximity. (C) NuFF cells were infected with AD169-GFP (MOI = 1, 72 hpi) and stained with antibodies against CXCR4 and US27 (left) or CXCR4 and US28 (right) followed by DuoLink PLA. Infected cells are shown in green, and red indicates PLA spots. Images were captured using a Zeiss LSM700 laser scanning confocal microscope, and representative images are shown. For PLA (B, C), images are maximum-intensity projections from z-stacks.
    Goat Anti Mouse Fitc, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cross adsorbed goat anti mouse igg conjugated to alexa fluor 568
    Confocal microscopy and adherence-invasion assays with pharmacologic inhibitors. Lipid raft-independent invasion of bronchial epithelial cells by NTHI. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI does not colocalize with vesicles positive for the following markers of lipid rafts: caveolin-1 (shown at 24 h), flotillin-1 (shown at 24 h), and cholesterol (shown at 4 h). NTHI was labeled with anti-11P6H antibody conjugated to <t>Alexa</t> Fluor 488 (green). Caveolin-1 was labeled with anti-caveolin-1 antibody and goat anti-mouse antibody conjugated to Alexa <t>Fluor</t> 568 (red). Flotillin-1 was labeled with anti-flotillin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Cholesterol was labeled with filipin (blue), and those samples were also labeled with anti-human secretory component antibody and donkey anti-goat antibody conjugated to Alexa Fluor 568 (red) to provide additional visualization of the plasma membrane. (B) Confocal microscopy. Filipin (FLP) and nystatin (NST) inhibit the internalization of fluorescently conjugated cholera toxin B subunit (white arrows), a known cargo of lipid raft-mediated endocytosis. H292 cells were pretreated with sRPMI containing inhibitor or inhibitor diluent, followed by addition of the cargo conjugate, incubation for 20 min on ice, and incubation for 2 h at 37°C. (C) Adherence-invasion assays. Filipin and nystatin do not inhibit invasion by NTHI. H292 cells were pretreated with inhibitor or inhibitor diluent for 2 h, followed by a 4-h infection conducted according to the adherence-invasion assay protocol. Data are normalized to samples in the absence of inhibitor. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.
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    Validation of external S. aureus labeling. Adherent GM-MФs were either treated with cytochalasin D (to block uptake) or control prior to and during incubation with GFP- S. aureus RN6390 and incubated with lysostaphin (to degrade external S. aureus , eliminating external bacteria) or control following incubation with S. aureus . External S. aureus were labeled with an IgG 3 monoclonal mouse anti- S. aureus primary antibody and Texas-RedX-conjugated goat anti-mouse secondary antibody. (A) No treatment. (B) Treatment with cytochalasin D. (C) Treatment with lysostaphin. (D) Treatment with both cytochalasin D and lysostaphin. (Original magnification, 200X).

    Journal: PLoS ONE

    Article Title: Heterogeneity in Macrophage Phagocytosis of Staphylococcus aureus Strains: High-Throughput Scanning Cytometry-Based Analysis

    doi: 10.1371/journal.pone.0006209

    Figure Lengend Snippet: Validation of external S. aureus labeling. Adherent GM-MФs were either treated with cytochalasin D (to block uptake) or control prior to and during incubation with GFP- S. aureus RN6390 and incubated with lysostaphin (to degrade external S. aureus , eliminating external bacteria) or control following incubation with S. aureus . External S. aureus were labeled with an IgG 3 monoclonal mouse anti- S. aureus primary antibody and Texas-RedX-conjugated goat anti-mouse secondary antibody. (A) No treatment. (B) Treatment with cytochalasin D. (C) Treatment with lysostaphin. (D) Treatment with both cytochalasin D and lysostaphin. (Original magnification, 200X).

    Article Snippet: To achieve red fluorescent labeling of external bacteria cells were incubated for 15 minutes with PBS/4% FBS (blocking buffer), incubated for 40 minutes with 10 µg/ml mouse monoclonal IgG3 anti-S. aureus antibody in blocking buffer, washed three times with blocking buffer, incubated for 20 minutes with 40 µg/ml Texas-RedX-conjugated goat anti-mouse IgG (Life Technologies) in blocking buffer, and washed twice with blocking buffer.

    Techniques: Labeling, Blocking Assay, Incubation

    Scanning cytometry fluorescence imaging with GFP-S. aureus RN6390. Adherent GM-MФs were incubated with unopsonized GFP- S. aureus RN6390. External S. aureus were labeled with an IgG 3 monoclonal mouse anti- S. aureus primary antibody and Texas-RedX-conjugated goat anti-mouse secondary antibody. Collapsed confocal stack images were acquired by scanning cytometry. (A) CellTracker Blue and Hoechst channel (cells). (B) GFP channel (all bacteria). (C) Texas Red channel (external bacteria). (D) Composite image. (Original magnification, 200X).

    Journal: PLoS ONE

    Article Title: Heterogeneity in Macrophage Phagocytosis of Staphylococcus aureus Strains: High-Throughput Scanning Cytometry-Based Analysis

    doi: 10.1371/journal.pone.0006209

    Figure Lengend Snippet: Scanning cytometry fluorescence imaging with GFP-S. aureus RN6390. Adherent GM-MФs were incubated with unopsonized GFP- S. aureus RN6390. External S. aureus were labeled with an IgG 3 monoclonal mouse anti- S. aureus primary antibody and Texas-RedX-conjugated goat anti-mouse secondary antibody. Collapsed confocal stack images were acquired by scanning cytometry. (A) CellTracker Blue and Hoechst channel (cells). (B) GFP channel (all bacteria). (C) Texas Red channel (external bacteria). (D) Composite image. (Original magnification, 200X).

    Article Snippet: To achieve red fluorescent labeling of external bacteria cells were incubated for 15 minutes with PBS/4% FBS (blocking buffer), incubated for 40 minutes with 10 µg/ml mouse monoclonal IgG3 anti-S. aureus antibody in blocking buffer, washed three times with blocking buffer, incubated for 20 minutes with 40 µg/ml Texas-RedX-conjugated goat anti-mouse IgG (Life Technologies) in blocking buffer, and washed twice with blocking buffer.

    Techniques: Cytometry, Fluorescence, Imaging, Incubation, Labeling

    Expression analysis of Wnt/β-catenin target genes, CD44 and EphB2, in the gastrointestinal tract of Ad Dkk1- or Ad Fc-treated adult C57BL/6 mice (12–16 weeks old). Organs were harvested 2 days after Ad Dkk1 i.v injection (10 9 pfu). ( Left ) Ad Dkk1 repression of CD44 expression in proliferative zones of all levels of the gastrointestinal epithelium. Arrowheads indicate the absence of CD44 immunoreactivity in proliferative compartments of the intestinal epithelium in Ad Dkk1 animals. * , residual CD44 staining in nonepithelial lamina propria. ( Right ) Ad Dkk1 repression of EphB2 in small intestine and colon. Repression was weaker in ascending colon and no repression was observed in stomach. EphB2 immunofluorescence was performed with Alexa 488 detection of EphB2 immunoreactivity (green) and Hoechst 33342 nuclear counterstain (blue). Stomach (st), duodenum (du), jejunum (je), ileum (il), cecum (ce), ascending colon (ac), and descending colon (dc) are shown.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1

    doi: 10.1073/pnas.2536800100

    Figure Lengend Snippet: Expression analysis of Wnt/β-catenin target genes, CD44 and EphB2, in the gastrointestinal tract of Ad Dkk1- or Ad Fc-treated adult C57BL/6 mice (12–16 weeks old). Organs were harvested 2 days after Ad Dkk1 i.v injection (10 9 pfu). ( Left ) Ad Dkk1 repression of CD44 expression in proliferative zones of all levels of the gastrointestinal epithelium. Arrowheads indicate the absence of CD44 immunoreactivity in proliferative compartments of the intestinal epithelium in Ad Dkk1 animals. * , residual CD44 staining in nonepithelial lamina propria. ( Right ) Ad Dkk1 repression of EphB2 in small intestine and colon. Repression was weaker in ascending colon and no repression was observed in stomach. EphB2 immunofluorescence was performed with Alexa 488 detection of EphB2 immunoreactivity (green) and Hoechst 33342 nuclear counterstain (blue). Stomach (st), duodenum (du), jejunum (je), ileum (il), cecum (ce), ascending colon (ac), and descending colon (dc) are shown.

    Article Snippet: Stainings were visualized with Alexa 488-conjugated secondary anti-goat antibodies (Molecular Probes) and nuclei were counterstained with Hoechst 33342 (Molecular Probes).

    Techniques: Expressing, Mouse Assay, Injection, Staining, Immunofluorescence

    US27 is found in close proximity to CXCR4. (A) Immunofluorescence microscopy of HEK293-US27 or HEK293-US28 cells stained with anti-FLAG (green) and anti-CXCR4 (red) followed by goat anti-mouse FITC and donkey anti-goat TRITC secondary antibodies, respectively. Nuclei were visualized with DAPI (blue). Bar, 10 μm. (B) HEK293-US27 or HEK293-US28 cells were stained with anti-FLAG and anti-CXCR4 followed by oligonucleotide-conjugated secondary antibodies and amplification using the DuoLink kit for the proximity ligation assay (PLA). Red PLA spots indicate receptors in close proximity. (C) NuFF cells were infected with AD169-GFP (MOI = 1, 72 hpi) and stained with antibodies against CXCR4 and US27 (left) or CXCR4 and US28 (right) followed by DuoLink PLA. Infected cells are shown in green, and red indicates PLA spots. Images were captured using a Zeiss LSM700 laser scanning confocal microscope, and representative images are shown. For PLA (B, C), images are maximum-intensity projections from z-stacks.

    Journal: Journal of Virology

    Article Title: Human Cytomegalovirus UL111A and US27 Gene Products Enhance the CXCL12/CXCR4 Signaling Axis via Distinct Mechanisms

    doi: 10.1128/JVI.01981-17

    Figure Lengend Snippet: US27 is found in close proximity to CXCR4. (A) Immunofluorescence microscopy of HEK293-US27 or HEK293-US28 cells stained with anti-FLAG (green) and anti-CXCR4 (red) followed by goat anti-mouse FITC and donkey anti-goat TRITC secondary antibodies, respectively. Nuclei were visualized with DAPI (blue). Bar, 10 μm. (B) HEK293-US27 or HEK293-US28 cells were stained with anti-FLAG and anti-CXCR4 followed by oligonucleotide-conjugated secondary antibodies and amplification using the DuoLink kit for the proximity ligation assay (PLA). Red PLA spots indicate receptors in close proximity. (C) NuFF cells were infected with AD169-GFP (MOI = 1, 72 hpi) and stained with antibodies against CXCR4 and US27 (left) or CXCR4 and US28 (right) followed by DuoLink PLA. Infected cells are shown in green, and red indicates PLA spots. Images were captured using a Zeiss LSM700 laser scanning confocal microscope, and representative images are shown. For PLA (B, C), images are maximum-intensity projections from z-stacks.

    Article Snippet: For standard immunofluorescence (IF), cells were costained with anti-CXCR4 goat polyclonal (1:100) and anti-FLAG mouse monoclonal antibody (1:500) followed by goat anti-mouse FITC and donkey anti-goat tetramethylrhodamine (TRTIC; 1:250), and then Prolong Gold mounting medium with DAPI (4′,6-diamidino-2-phenylindole; Invitrogen) was added.

    Techniques: Immunofluorescence, Microscopy, Staining, Amplification, Proximity Ligation Assay, Infection

    cmvIL-10 enhances CXCR4 signaling in HCMV-infected cells. (A) NuFF cells were infected with TB40/E- mCherry (MOI = 1). Total RNA was harvested from mock- or HCMV-infected NuFFs at 72 h postinfection (hpi) and reverse transcribed, and UL123 (IE1) or β-actin genes were amplified. The resulting bands were visualized via agarose gel electrophoresis. (B) Mock- or HCMV-infected NuFF cells were stained with IL-10R-FITC or isotype control antibody and analyzed by flow cytometry. (C) Flow cytometric comparison of mock- or HCMV-infected NuFF cells for mCherry fluorescence as a measure of infection (left) or IL-10R stained as described above (right). (D) Calcium response was evaluated in Fluo-4 AM-loaded mock- or HCMV-infected cells stimulated with 100 ng/ml CXCL12 ± 100 ng/ml cmvIL-10 at 72 hpi. Relative fluorescence units (RFU) were measured by flow cytometry; the arrow indicates stimulus addition. (E) Transwell migration of mock-infected (gray bars) or HCMV-infected (black bars) NuFF cells toward CXCL12 in the lower chamber ± 100 ng/ml cmvIL-10 (striped bars, mock infected; white bars, HCMV infected) was measured at 72 hpi. Error bars, standard error for 3 replicates; *, P

    Journal: Journal of Virology

    Article Title: Human Cytomegalovirus UL111A and US27 Gene Products Enhance the CXCL12/CXCR4 Signaling Axis via Distinct Mechanisms

    doi: 10.1128/JVI.01981-17

    Figure Lengend Snippet: cmvIL-10 enhances CXCR4 signaling in HCMV-infected cells. (A) NuFF cells were infected with TB40/E- mCherry (MOI = 1). Total RNA was harvested from mock- or HCMV-infected NuFFs at 72 h postinfection (hpi) and reverse transcribed, and UL123 (IE1) or β-actin genes were amplified. The resulting bands were visualized via agarose gel electrophoresis. (B) Mock- or HCMV-infected NuFF cells were stained with IL-10R-FITC or isotype control antibody and analyzed by flow cytometry. (C) Flow cytometric comparison of mock- or HCMV-infected NuFF cells for mCherry fluorescence as a measure of infection (left) or IL-10R stained as described above (right). (D) Calcium response was evaluated in Fluo-4 AM-loaded mock- or HCMV-infected cells stimulated with 100 ng/ml CXCL12 ± 100 ng/ml cmvIL-10 at 72 hpi. Relative fluorescence units (RFU) were measured by flow cytometry; the arrow indicates stimulus addition. (E) Transwell migration of mock-infected (gray bars) or HCMV-infected (black bars) NuFF cells toward CXCL12 in the lower chamber ± 100 ng/ml cmvIL-10 (striped bars, mock infected; white bars, HCMV infected) was measured at 72 hpi. Error bars, standard error for 3 replicates; *, P

    Article Snippet: For standard immunofluorescence (IF), cells were costained with anti-CXCR4 goat polyclonal (1:100) and anti-FLAG mouse monoclonal antibody (1:500) followed by goat anti-mouse FITC and donkey anti-goat tetramethylrhodamine (TRTIC; 1:250), and then Prolong Gold mounting medium with DAPI (4′,6-diamidino-2-phenylindole; Invitrogen) was added.

    Techniques: Infection, Amplification, Agarose Gel Electrophoresis, Staining, Flow Cytometry, Cytometry, Fluorescence, Migration

    Confocal microscopy and adherence-invasion assays with pharmacologic inhibitors. Lipid raft-independent invasion of bronchial epithelial cells by NTHI. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI does not colocalize with vesicles positive for the following markers of lipid rafts: caveolin-1 (shown at 24 h), flotillin-1 (shown at 24 h), and cholesterol (shown at 4 h). NTHI was labeled with anti-11P6H antibody conjugated to Alexa Fluor 488 (green). Caveolin-1 was labeled with anti-caveolin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Flotillin-1 was labeled with anti-flotillin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Cholesterol was labeled with filipin (blue), and those samples were also labeled with anti-human secretory component antibody and donkey anti-goat antibody conjugated to Alexa Fluor 568 (red) to provide additional visualization of the plasma membrane. (B) Confocal microscopy. Filipin (FLP) and nystatin (NST) inhibit the internalization of fluorescently conjugated cholera toxin B subunit (white arrows), a known cargo of lipid raft-mediated endocytosis. H292 cells were pretreated with sRPMI containing inhibitor or inhibitor diluent, followed by addition of the cargo conjugate, incubation for 20 min on ice, and incubation for 2 h at 37°C. (C) Adherence-invasion assays. Filipin and nystatin do not inhibit invasion by NTHI. H292 cells were pretreated with inhibitor or inhibitor diluent for 2 h, followed by a 4-h infection conducted according to the adherence-invasion assay protocol. Data are normalized to samples in the absence of inhibitor. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.

    Journal: Infection and Immunity

    Article Title: Internalization and Trafficking of Nontypeable Haemophilus influenzae in Human Respiratory Epithelial Cells and Roles of IgA1 Proteases for Optimal Invasion and Persistence

    doi: 10.1128/IAI.00864-13

    Figure Lengend Snippet: Confocal microscopy and adherence-invasion assays with pharmacologic inhibitors. Lipid raft-independent invasion of bronchial epithelial cells by NTHI. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI does not colocalize with vesicles positive for the following markers of lipid rafts: caveolin-1 (shown at 24 h), flotillin-1 (shown at 24 h), and cholesterol (shown at 4 h). NTHI was labeled with anti-11P6H antibody conjugated to Alexa Fluor 488 (green). Caveolin-1 was labeled with anti-caveolin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Flotillin-1 was labeled with anti-flotillin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Cholesterol was labeled with filipin (blue), and those samples were also labeled with anti-human secretory component antibody and donkey anti-goat antibody conjugated to Alexa Fluor 568 (red) to provide additional visualization of the plasma membrane. (B) Confocal microscopy. Filipin (FLP) and nystatin (NST) inhibit the internalization of fluorescently conjugated cholera toxin B subunit (white arrows), a known cargo of lipid raft-mediated endocytosis. H292 cells were pretreated with sRPMI containing inhibitor or inhibitor diluent, followed by addition of the cargo conjugate, incubation for 20 min on ice, and incubation for 2 h at 37°C. (C) Adherence-invasion assays. Filipin and nystatin do not inhibit invasion by NTHI. H292 cells were pretreated with inhibitor or inhibitor diluent for 2 h, followed by a 4-h infection conducted according to the adherence-invasion assay protocol. Data are normalized to samples in the absence of inhibitor. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.

    Article Snippet: Host cell components were visualized using filipin III fluorescent dye (final concentration of 333 μg/ml; Cayman Chemical), primary monoclonal antibodies including mouse anti-caveolin-1 antibody (1:50; BD), mouse anti-early endosomal antigen 1 (EEA-1) antibody (1:200; BD), mouse anti-flotillin-1 antibody (1:20; BD), mouse anti-human CD107a antibody (LAMP1) (1:200; BD), and goat anti-human secretory component antibody (1:200; Sigma), and secondary antibodies including highly cross-adsorbed goat anti-mouse IgG conjugated to Alexa Fluor 568 (Life Technologies, 1:200) and donkey anti-goat IgG conjugated to Alexa Fluor 568 (Life Technologies, 1:200).

    Techniques: Confocal Microscopy, Labeling, Incubation, Infection, Invasion Assay

    Confocal microscopy and survival assay with pharmacologic inhibitor of lysosome acidification. NTHI traffics to early endosomes and lysosomes and is killed in lysosomes. IgA1 proteases are required for optimal survival in lysosomes. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI is found within vesicles positive for EEA1 (shown at 4 h) and within vesicles positive for LAMP1 (shown at 24 h). NTHI was labeled using anti-11P6H antibody conjugated to Alexa Fluor 488 (green). EEA1 was labeled using anti-EEA1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). LAMP1 was labeled using anti-human LAMP1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). (B) Survival assay in the presence and absence of concanamycin A (CMA). H292 cells were infected for 16 h, treated with gentamicin for 1 h, and treated with concanamycin or diluent for 3 h. Survival data are normalized to samples treated with diluent. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.

    Journal: Infection and Immunity

    Article Title: Internalization and Trafficking of Nontypeable Haemophilus influenzae in Human Respiratory Epithelial Cells and Roles of IgA1 Proteases for Optimal Invasion and Persistence

    doi: 10.1128/IAI.00864-13

    Figure Lengend Snippet: Confocal microscopy and survival assay with pharmacologic inhibitor of lysosome acidification. NTHI traffics to early endosomes and lysosomes and is killed in lysosomes. IgA1 proteases are required for optimal survival in lysosomes. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI is found within vesicles positive for EEA1 (shown at 4 h) and within vesicles positive for LAMP1 (shown at 24 h). NTHI was labeled using anti-11P6H antibody conjugated to Alexa Fluor 488 (green). EEA1 was labeled using anti-EEA1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). LAMP1 was labeled using anti-human LAMP1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). (B) Survival assay in the presence and absence of concanamycin A (CMA). H292 cells were infected for 16 h, treated with gentamicin for 1 h, and treated with concanamycin or diluent for 3 h. Survival data are normalized to samples treated with diluent. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.

    Article Snippet: Host cell components were visualized using filipin III fluorescent dye (final concentration of 333 μg/ml; Cayman Chemical), primary monoclonal antibodies including mouse anti-caveolin-1 antibody (1:50; BD), mouse anti-early endosomal antigen 1 (EEA-1) antibody (1:200; BD), mouse anti-flotillin-1 antibody (1:20; BD), mouse anti-human CD107a antibody (LAMP1) (1:200; BD), and goat anti-human secretory component antibody (1:200; Sigma), and secondary antibodies including highly cross-adsorbed goat anti-mouse IgG conjugated to Alexa Fluor 568 (Life Technologies, 1:200) and donkey anti-goat IgG conjugated to Alexa Fluor 568 (Life Technologies, 1:200).

    Techniques: Confocal Microscopy, Clonogenic Cell Survival Assay, Labeling, Infection