Triton x

JAWS II and GM-BM cells were seeded on coverslips placed in a 24-well plate at a density of 1.5 × 10⁵ cells per well. Cells were left uninfected or were treated with ECTV for 60 min. at 37 °C. After 4, 12 and 24 h, cells were fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich, St Louis, MO, USA) and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) in PBS. Next, JAWS II and GM-BM cells were blocked with 3% bovine serum albumin (BSA, Sigma-Aldrich) in 0.1% Triton X-100 and incubated for 45 min. with anti-cathepsin B, anti-cathepsin L (both from Abcam, Cambridge, MA, USA) and anti-cystatin B (Thermo Fisher Scientific) primary antibodies. After washing with 0.1% Triton X-100 in PBS, cells were incubated with secondary anti-mouse or anti-rabbit antibodies conjugated with rhodamine Red-X (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) diluted in blocking solution for 1 h. ECTV antigens were stained with FITC-conjugated polyclonal antibodies for 1 h. Viral and nuclear DNA was stained with Hoechst 33342 (Sigma-Aldrich) solution for 10 min. in the dark. Slides were mounted in ProLong Gold Antifade Reagent (Life Technologies). Images were captured using Olympus BX60 fluorescence microscope and analyzed with Cell^F software (Soft Imaging System, Olympus, Tokyo, Japan) and ImageJ (NIH, Bathesda, MD, USA).

Cells were rinsed once with PBS, fixed in 4% paraformaldehyde for 30 min, and rinsed three times with PBS at room temperature. A blocking solution was made with 0.1% Triton-X and 5% normal donkey serum (Jackson ImmunoResearch, West Grove, PA) in PBS, and cells were incubated for 1 hour at room temperature before staining with primary antibodies in a blocking solution at 4°C overnight. The cells were then washed three times in PBS 0, 1% triton before being incubated for 2 h at room temperature with secondary antibodies, and 0.1 mg/ml DAPI (Invitrogen, Carlsbad, CA) in a blocking solution for 1 h. Finally, cells were washed three times in PBS 0, 1% triton and mounted using Vectashield Antifade Mounting Medium (Vector Laboratories, Burlingame, CA). All primary and secondary antibodies used are listed in Table 1.
Fluorescence images were acquired using a confocal TCS SP5 microscope (Leica Microsystems). Image processing was performed with ImageJ software (NIH).