Rabbit anti ad5

Cells were fixed for 20 min with 4 % PFA in PBS at room temperature or for 5 min with −20 °C methanol on ice. Cells were permeabilized and blocked with 5 % normal goat serum (Invitrogen, Carlsbad, CA, USA) and 0.5 % Triton X-100 (Sigma Aldrich, St. Louis, MO, USA) in PBS for 1 h at room temperature. Primary antibody labeling was performed for 1 h at room temperature using rabbit anti-Cx43 (1:2000; Sigma Aldrich, St. Louis, MO, USA), mouse anti-E2A [B6–8] (1:250; generously provided by D. Ornelles, Wake Forest School of Medicine, Microbiology and Immunology), mouse anti-Adenovirus E1A [M73] (1:500; Abcam, Cambridge, UK), mouse anti-β-catenin (1:50; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-Ad5 (1:5000; Abcam, Cambridge, UK), and mouse anti-ZO-1(1:500; BD Biosciences, San Jose, CA, USA). Cells were washed 6 times prior to secondary antibody labeling for 1 h at room temperature with goat secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 555 (Thermo Fisher, Waltham, MA, USA). During secondary antibody labeling cells were counterstained with DAPI and wheat germ agglutinin (WGA) conjugated Alexa Fluor 647. Slides were mounted using Prolong Gold Antifade (Life Technologies, Carlsbad, CA, USA).