Fitc labeled secondary antibody

Cells grown on membrane filters and PCLS were fixed for 20 min at room-temperature with 3% paraformaldehyde in PBS. Fixed cells were washed twice with 0.1 M glycine in PBS and then permeabilized with 0.2% Triton X-100 diluted in PBS for 10 min. All antibodies were diluted in PBS containing 1% bovine serum albumin at room temperature. BPIV3-infected cells were stained with a polyclonal antiviral antiserum (VMRD, caprine origin) followed by a FITC-labeled secondary antibody (Sigma-Aldrich). Cilia were stained by using a Cy3-labeled monoclonal antibody against β-tubulin (Sigma-Aldrich). Basal cells were visualized by a monoclonal mouse anti-human CD142 antibody (Serotec) and a primary antibody specific for TTF-1 (clone BGX-397A mouse monoclonal, BioGenex) was used to stain pneumocytes type II. Nuclei were visualized with DAPI (4′, 6′-diamidino-2-phenylindole) which was added to the apical surface of the cells and removed after incubation for 15 min (37 °C) followed by three washing steps with PBS. After washing, cells were embedded in Mowiol resin and photomicrographs were generated using a Leica TCS SP5 AOBS confocal laser scanning microscope. For image processing, Adobe Photoshop (Version 10.0, Adobe Systems, USA) was used.

For MRP1 and BCRP (BXP-21 clone) staining cells were permeabilized using the Fixation/Permeabilization Solution Kit (BD Biosciences, USA). For other stainings cells were detached using NonEnzymatic Cell Dissociation Solution (ATCC, USA). Next all cells were labeled with antibodies specific for CD243 (anti-MDR1/1, anti-MDR1/2 and anti-MDR1/3); MRP1 (anti-MRP1/1 and anti-MRP1/2); MRP4 (anti-MRP4/1 and anti-MRP4/2); MRP5 (anti-MRP5/1); BCRP (anti-BCRP/1 and anti-BCRP/2) and the appropriate isotypic control for 30 min at 4°C (Table 4).
After washing with PBS cells were analyzed or detection by incubation with the corresponding FITC-labeled secondary antibody (Sigma Aldrich, USA) was performed for an additional 30 min at 4°C. After washing with PBS cells were analyzed using a FACSCalibur flow cytometer, and data were processed using CellQuest software (BD Biosciences, USA) for 3 independent experiments. MDR protein expression was evaluated using the Kolmogorov-Smirnov statistic automatically calculated by CellQuest software (D value ≥0.2 was evaluated as positive). The concentrations of used antibodies are presented in Table 5.

After indicated doses of treatment, IK cells were maintained at 37°C and 5%CO2 until fixation at 24 hours post-treatment. Cells were washed twice with phosphate-buffered saline (PBS), fixed for 20 min with 4% paraformaldehyde, permeabilized with methanol during 5 min, washed and maintained with PBS. After blocking (5% HS +5% FBS +0,2% Glicina +0,1% Tritó in PBS) during 1 h, cells were incubated overnight at 4°C with a monoclonal anti-γH2AX antibody (monoclonal, Millipore), anti-Acetyl-Histone H4 (Lys12) (polyclonal, Cell-Signal) 1∶500 in PBS, and washed and incubated with FITClabeled secondary antibody (Sigma) 1∶500 in PBS in the dark for 1 h at room temperature. Cells were then washed, counterstained, and stained with bis-benzimide fluorescent dye (Hoechst 33258), to a final concentration of 0.5 mg/ml, in the dark for 35 minutes. Cells were counted under epifluorescence microscope (Leica Microsystems, Wetzlar, Germany). Aleatory sampling methods were used to select the images and all the cells in each selected image were screened. The γH2AX foci per nucleus were counted by eye. Cells were judged as “positive” for γH2AX foci if they displayed 10 or more discrete dots of brightness. For quantitation of foci, a minimum of 300 cells were analysed for each time point.

After 4 days of culture, OECs seeded on Ti plates were fixed with 4% paraformaldehyde for 10 min and pretreated with 0.5% Triton X-100 (Novocastra Laboratories, Newcastle-upon-Tyne, UK) for 3 min. The samples were then incubated with a polyclonal mouse anti-rat Ln-332 antibody (1:100 dilution, Santa Cruz Biotechnology, CA, USA) overnight at 4 °C. Samples were subsequently incubated with a fluorescein isothiocyanate (FITC)-labeled secondary antibody (1:100 dilution; Chemicon International, Billerica, MA, USA) at 37 °C for 2 h. Actin filaments were stained with tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin (1:100 dilution, Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 1 h. Imaging was performed using fluorescence microscopy (BZ-9000; Keyence, Osaka, Japan).