Alexa fluor 555 conjugated anti mouse antibody

Cells were seeded on to glass coverslips in 24-well plates at 10,000/cm² and exposed to different concentrations of ribavirin analogs or ribavirin (20 or 50 μg/mL) for 1, 3 and 7 days of culture. At each time-point, cells were fixed with 3% paraformaldehyde (PFA) in PBS and washed with PBS containing 0.5% BSA. They were permeabilized with 0.1% Triton X100 in PBS and incubated for 2 h at room temperature with anti-αv integrins (sc-9969, Santa-Cruz) or anti-eIF4E (610270, BD PharMingen) antibodies. After washing, the coverslips were incubated with the appropriate fluorescent secondary antibodies: Alexa Fluor 555-conjugated anti-mouse antibody (A21424, Invitrogen) or Alexa Fluor 488-conjugated anti-mouse antibody (BD Transduction Laboratories). The actin cytoskeleton was stained with FITC- or TRITC-phalloidin (P5282 or P1981, Sigma Aldrich). For each assay, cell nuclei were stained with DAPI (4,6-diamidino-2-phenylindole dihydrochloride, D9542, Sigma Aldrich). Coverslips were mounted in Prolong-Gold Antifade Reagent (P36930, Invitrogen) and examined by laser scanning confocal microscopy (LSM710, Zeiss). Controls, in which primary antibodies were replaced with PBS, were negative. Fluorescence microscopy figures were processed using Fiji software [17 (link)].

Cells were seeded on glass coverslips and after treatment cells were washed and fixed with ice-cold methanol. The samples were blocked with 10% normal goat serum for 1 h and incubated with phospho-histone-γ-H2AX antibody (Ser139; Upstate Biotechnology, Temecula, CA, USA) and p53-binding protein 1 (53BP1) antibody (Cell Signaling). Samples were washed three times for 5 min in PBS, and then incubated with Alexa Fluor 488-conjugated anti-rabbit antibody and Alexa Fluor 555-conjugated anti-mouse antibody (Invitrogen, Carlsbad, CA, USA) for 1 h. Nuclei were counterstained with DAPI contained in Vecatshield mounting medium (Vector Laboratories Inc, Burlingame, CA, USA). The stained cells were then analyzed under a fluorescence microscope (Axio Imager M2, Carl Zeiss, Thornwood, NY, USA) with a × 63 objective (oil immersion, aperture 1.3) with five slices of z-stacks of 0.2 μM thickness each. Quantitative image analysis of 40 nuclei from each experiment was performed using Cell module in Imaris software version 8.0 (Bitplane, Concord, MA, USA).

Cells (5 × 10⁴ cells/ml) cultured on glass coverslips in six-well plates were fixed with 4% paraformaldehyde and were incubated with the primary antibodies in PBS with 1% bovine serum albumin and 0.1% Triton X-100 at 4°C overnight. CD44 (Cell Signaling Technology, USA), β-catenin (Santa Cruz), Vimentin (Thermo Fisher Scientific), and TM4SF4 (Sigma-Aldrich) antibodies were used. Staining was visualized using an Alexa Fluor 488–conjugated anti-rabbit or Alexa Fluor 555–conjugated anti-mouse antibody (Invitrogen). Nuclei were counterstained using 4, 6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Stained cells were visualized with a fluorescence microscope (Olympus BX53F; Olympus, Tokyo, Japan).

All plasmids in this study were constructed and sequenced to confirm their identity. The Tim-4, Tim-4^AAA, Tim-4^TL, Tim-4-GPI, and Anxa5-GPI plasmid used in this study have been previously described^{33 (link)}. Tim-4^ΔIgV lacking the IgV domain of Tim-4 (residues 25–134) and Tim-4^ΔMucin without the mucin domain (residues 135–270) were constructed into the pDisplay vector using a PCR-based method. The antibodies used in the study were anti-HA antibody (Santa Cruz, SC-7392), PE-conjugated anti-F4/80 antibody (Biolegend, 123110), Alexa Fluor 405-conjugated anti-mouse antibody (Invitrogen, A31553), Alexa Fluor 488-conjugated anti-mouse antibody (Invitrogen, A-11029), and Alexa Fluor 555-conjugated anti-mouse antibody (Invitrogen, A-21422). Polystyrene beads (FluoSpheres carboxylate beads, Invitrogen, F8826), FITC-labeled E. coli (E-2861), S. aureus (S-2851), and zymosan A (Z-2841) bioparticles were purchased from Invitrogen. Alexa Fluor 488-conjugated LPS (L-23351) was purchased from Molecular Probes. LPS from E. coli O55:B5 (L2880) was purchased from Sigma Aldrich. About 6.0–8.0-μm streptavidin-coated polystyrene particles (SVP-60-5) were purchased from Spherotech. For IgG opsonization, FITC-conjugated streptavidin antibody (200-402-095) was purchased from Rockland Immunochemicals. Dexamethasone (265005) and TAMRA-SE (C1171) were purchased from Merck and ThermoFisher, respectively.

For the BrDU assay, cancer cells were seeded on to glass coverslips in 24-well plates at 10,000/cm² and exposed to SRO-91 or ribavirin at 0, 20 or 50 μg/mL for 7 days of culture. After each time-point, the medium was supplemented with BrDU (40 μM, B9285, Sigma) for 3 h before the cells were washed with PBS. They were fixed with 3% paraformaldehyde and incubated for 30 min with HCl (2N) (LCH, Chimie, Les Aires France) to denature DNA. Cells were incubated with borate salt (0.1 M, Sigma) to neutralize HCl before being permeabilized with 0.1% Triton X100 in PBS. Cells were incubated for 2 h at room temperature with anti-BrDU (B2531, Sigma) antibody. After washing, the coverslips were incubated with fluorescent secondary antibodies Alexa Fluor 555-conjugated anti-mouse antibody (A21424, Invitrogen). Cell nuclei were stained with DAPI (4,6-diamidino-2-phenylindole dihydrochloride, D9542, Sigma Aldrich). Controls, in which primary antibodies were replaced with PBS, were negative. Coverslips were mounted in Prolong Gold antifade Reagent and examined with laser scanning confocal microscopy with a mosaic of 4x4 independent fields. Positive BrDU (cells/field) were quantified with the software ImageJ ® (National Institutes of Health, Bethesda, MD).