Ultracruz mounting medium

A 10⁴ aliquot of BHK-21 cells was grown on glass coverslips coated with 50 μg/ml poly(D) lysine (Sigma-Aldrich) in 35 mm tissue culture plates for immunostaining detection of viral protein localization in cells. Twenty-four hours after seeding, the cells were rinsed with PBS and infected with 1 ml of cell-free culture supernatant for 6 h. After the infected cells were incubated for the desired length of time, they were rinsed once with PBS and then fixed in 3% paraformaldehyde for 10 minutes at room temperature. After rinsing with PBS-GSA (10 mM glycine and 0.2% sodium azide), the cells were permeabilized with 0.5% Triton-X-100 for exactly 3 min. After blocking (3% bovine serum albumin in PBS-GSA), coverslips were successively incubated with rabbit polyclonal anti-PFV-IN (1:40) or rabbit polyclonal anti-PFV-Gag (1:150) for 1 h. Cells were then washed and incubated for 30 min with 1:80 (PFV-IN) or 1: 200 (PFV-Gag) dilutions of FITC-conjugated donkey anti-rabbit IgG (sc-2089, Santa Cruz Biotechnology Inc., USA) secondary antibody. Finally, the coverslips were mounted onto a microslide using a drop of UltraCruz mounting medium (sc-24941, Santa Cruz Biotechnology) with DAPI. Fluorescence microscopy observations were performed with a fluorescence microscope (Nikon, Eclipse, TE 2000-U, Japan) at a magnification of 400×.

Normal human epidermal keratinocytes (2 × 10⁴) cultured on slides (Lab-Tek, Rochester, NY, USA) with or without Gly were washed in phosphate-buffered saline (PBS), fixed with acetone for 10 min and blocked using 10% bovine serum albumin in PBS for 30 min. Samples were incubated with primary rabbit anti-AhR or Nrf2 (1:50) in western breeze blocker diluent (Invitrogen, Carlsbad, CA, USA) overnight at 4°C. Slides were washed with PBS before incubation with antirabbit secondary antibody (Alexa Fluor 546 or 488; Molecular Probes, Eugene, OR, USA) for 1 h at room temperature. After nuclear staining with 4′,6-diamidino-2-phenylindole, slides were mounted with UltraCruz mounting medium (Santa Cruz Biotechnology). All samples were analyzed using a D-Eclipse confocal laser scanning microscope (Nikon, Tokyo, Japan).

SSTR2 levels in all three cell lines were determined by flow cytometry and immunofluorescence. 10⁵ cells/well were seeded in either 8-well chamber slides (Lab-Tek, immunofluorescence) or 24-well plates (Corning, flow cytometry). The medium was removed and the cells were washed twice with phosphate buffered saline (PBS, 500 µL). Then bovine serum albumin (2% BSA, 500 µL) was added and the cells were incubated for 1 h at 4 °C, followed by addition of anti-SSTR2 monoclonal antibody or isotype control (R&D Systems, 1:100 dilution). The cells, with the antibody, were incubated for an additional 1 h at room temperature followed by three PBS washes. Subsequently, goat anti-mouse antibody (R&D Systems, 500 µL, 1:100 in 2% BSA) was added and the cells were incubated for additional 1 h at room temperature, followed by three PBS washes. Cells were mounted with UltraCruz mounting medium containing DAPI (Santa Cruz Biotechnology, TX) and fluorescence images were determined by an epifluorescence microscope (200×; Olympus, X81) or a confocal microscope (ZEISS). For flow cytometry, the cells were quantitatively analyzed by LSR II (BD Biosciences) using FlowJo (Tristar).