Fluoview fv1000 confocal system

Cells (A498 or RAW264.7) were cultured on cell culture dishes, infected with bacterial strains for different time periods, washed, fixed for 15 min with 4% paraformaldehyde in PBS, permeabilized for 20 min in 0.1% Triton X-100 in PBS and blocked using 5% BSA for 1 h. Then, the cells were stained with the indicated corresponding primary antibodies and incubated with fluorescence-conjugated goat anti-mouse IgG (Invitrogen). The nuclei were counterstained with DAPI (Cell Signaling). The slides were mounted using Fluorescence Antifade Mountant (Molecular Probes). Images were captured at room temperature using a confocal microscope (Olympus FluoView FV1000 Confocal System) with a 63× oil immersion objective and Olympus FluoView software (Olympus). The confocal images shown are representative of three independent experiments.

Ready to use HEp-20-10 slides (Euroimmun; product No. FA 1512-20) were stained according to manufacturer’s instructions with minor modifications. Recombinant mAbs (mouse IgG1) were diluted to 5 µg/ml or 25 µg/ml in block buffer (0.1% tween, 0.5% BSA in PBS). After one hour of incubation with the mAbs, slides were washed in block buffer and stained with goat anti-mouse IgG-Alexa488 (Life Technologies) and 2 µg/ml DAPI (Sigma) for one hour. After washing, coverslips were mounted with Fluoro-gel (Electron Microscopy Sciences). Images were acquired using an Olympus Fluoview FV1000 confocal system.

VSMCs were seeded into a 24well plate at a density of 3 × 10⁴ cells/ml and cultured for 24 h. Cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.2% Triton X-100 for 10 min at RT. After washing, cells were blocked with 2% BSA in PBS for 1 h, removed from the blocking solution, and incubated overnight at 4 °C with rabbit anti-Nrf2 (1:50) (Cell Signaling, Beverly, MA, US), rabbit anti-AhR (1:100) (BioWord, MN, US), rabbit-anti-FAK (1:100) (Cell Signaling, Beverly, MA, US), mouse anti-paxillin (1:300) (Sigma, MO, US), mouse anti-integrin β1 (1:100) (Sigma, MO, US) and actin stains with DyLight™ 594 Phalloidin (1:20) (Cell Signaling, Beverly, MA, US). Then, the cells were gently washed under the cover slip three times with PBS and mounted in fluoroshield™ mounting medium with DAPI (Cell Signaling, Beverly, MA, US). Cells were visualized under a laser scanning confocal microscope (Fluoview FV1000 confocal system, Olympus). To ensure blinded data analysis, immunofluorescence staining were performed by one person, and all images and samples were recorded by another person.

IFAs were performed to detect viral antigens in T cells. Cells were harvested, washed twice with PBS, and suspended at 1 × 10⁶ cells/mL. The cells were mounted on gelatin-coated slides by liquid-based thin layer cytologic preparation; the slides were fixed with 4% paraformaldehyde for 25 min and permeabilized with 0.3% Triton-X100 for 30 min at room temperature. A blocking buffer containing 3% bovine serum albumin (Sigma) in PBS was used to block potential nonspecific binding sites at room temperature for 40 min. All slides were double-stained with anti-CD4 (Abcam) or CD8 (Abcam) antibodies and 1A8 to visualize HTNV-NP-positive CD4 or CD8 T cells, and fluorescence images were captured using the FLUOVIEW FV1000 confocal system (Olympus, Tokyo, Japan). Approximately 1000 cells from each group were examined, and cells displaying HTNV nucleocapsid protein were enumerated.

The fluorescence resonance energy transfer (FRET) was measured by the donor-sensitized acceptor fluorescence technique as described previously and calculated using custom Matlab scripts (Barda-Saad et al., 2005 (link); Braiman et al., 2006 (link); Jaron-Mendelson et al., 2012 (link)). Briefly, the ECFP excitation/EYFP emission, the ECFP excitation/ECFP emission, and the ECFP excitation/EYFP emission images were collected. Calibration curve of background is derived from single- and double-fluorescent protein samples. RETcorr is defined to be the pixel intensity in the corrected FRET image. Then, pixel-by-pixel calculation of FRET is carried out using the equation FRETeff = FRETcorr/(FRETcorr + ECFP) ×100%. Fluorescent images were acquired on a FluoView FV1000 confocal system (Olympus) using a 63 × /1.35 UPLSAPO objective (Olympus).