Immunocytochemistry of hCECs infected with CMV

Immunocytochemistry (ICC) was performed as previously described.³⁴ (link) The cells were grown in chamber slides. The hCECs were fixed in ice-cold 4% paraformaldehyde for 15 minutes 1 day after infection and then permeabilized with 0.3% Triton X-100 (Sigma-Aldrich) for 5 minutes and blocked in 3% BSA for 30 minutes. The primary antibodies were anti-CMV, clone 8B1.2 (1:2000; Sigma-Aldrich); anti-ZO-1 (1:100; Abcam, Cambridge, UK); anti-TNF-α (1:500; Abcam); and anti-nuclear factor kappa B (NF-κB) (1:100; Sigma-Aldrich). Alexa Fluor 488 and 594 secondary antibodies (1:1000) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). We further determined if changes induced by CMV infection were suppressed by ROCK inhibitors (Y27632, K115), because the Rho–ROCK pathway is a common downstream component of TNF-α signaling.