alexa fluor 488 goat anti mouse igg h l  (Thermo Fisher)

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

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

Journal: Infection and Immunity

Article Title: Inhibition of Adhesion of Escherichia coli K88ac Fimbria to Its Receptor, Intestinal Mucin-Type Glycoproteins, by a Monoclonal Antibody Directed against a Variable Domain of the Fimbria

doi:

Figure Lengend Snippet: Determination by Western blotting of the specificity of MAbs with regard to K88ab, K88ac, and K88ad adhesins. K88ab, K88ac, and K88ad adhesin antigens were separated by SDS-PAGE and transferred to nitrocellulose membranes. These membranes were probed with MAb 36/41 or 221/38 or BSA, followed by incubation with peroxidase-conjugated goat anti-mouse IgG as described in Materials and Methods. Molecular mass standards are indicated on the left. MAb 36/41 binds to K88ac fimbriae specifically, and MAb 221/38 binds to all three variants of K88 fimbriae.

Article Snippet: The plates were then washed as before, peroxidase-conjugated goat anti-mouse immunoglobulins (Pierce, Rockford, Ill.) were added, and the plates were again incubated.

Techniques: Western Blot, SDS Page, Incubation

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

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

Journal: Redox Biology

Article Title: Cx26 partial loss causes accelerated presbycusis by redox imbalance and dysregulation of Nfr2 pathway

doi: 10.1016/j.redox.2018.08.002

Figure Lengend Snippet: Decreased levels of endogenous antioxidant defenses in the cochlea of Gjb2 +/− mice. A: Western blot analysis of Nrf2 and HO-1 expression in Gjb2 lox P /lox P and Gjb2 +/− cochleae (n = 8 for each condition) at 2, 6 and 12 months of age (M). B, C: Histograms (mean ± s.e.m.) represent optical density values normalized to GAPDH. Experiments were performed in triplicate and p-values were determined by two-tailed t -test. D: Immunofluorescence analysis of Nrf2 (upper panels, red fluorescence) and HO-1 expression (lower panels, green fluorescence) in the organ of Corti (oC), spiral ganglion neurons (SGNs) and stria vascularis (StV) at 6 months (M). Scale bars: oC, 20 µm; SGNs, 15 µm; StV, 50 µm.

Article Snippet: After washing in PBS, samples were incubated at room temperature for 2 h in labeled conjugated goat anti-rabbit (HO-1) (Alexa Fuor 488 Cat. No. A-11034, ThermoFischer, Waltham, MA, USA.) or donkey anti-mouse (GSH, Nrf2) secondary antibody (Alexa Fluor 488, Cat. No. A- 21202; or Alexa Fluor 546, IgG, Cat. No. A 10036, ThermoFischer) diluted 1:400 in 0.1 M PBS and stained with DAPI stained (1:500 in 0.1 M PBS).

Techniques: Mouse Assay, Western Blot, Expressing, Two Tailed Test, Immunofluorescence, Fluorescence