Fitc anti mouse antibody

GIPZ control and shIFN-β cells were seeded into 96-well plate (1 × 10⁴ cells/well) and pretreated with MDP for 72 h followed by infection with a clinical isolate of HCMV at MOI 1. Twenty-four h after infection, cells were fixed with methanol/acetone (1:1) at −20 °C for 10 min. Cells were then blocked with 7.5% BSA for 30 min. and incubated for 1 h with anti-human CMV IE1&2 antibody (MAB810) (1:500 in 0.5% BSA). After 3 washes with PBS, cells were incubated with FITC-anti mouse antibody (Sigma, 1:500, in 0.5% BSA) for 1 h. Then cells were washed 3 times with PBS and counterstained with propidium iodide (Invitrogen) for nuclear staining. Images were taken using Nikon Eclipse TS100 microscope.

For immunofluorescence analyses, cells were washed three times with PBS and fixed 10 min with ice-cold acetone-methanol (1:1) at room temperature. After three washes, cells were permeabilized with 0.25% Triton X-100 in PBS for 5 min and incubated 1 h with the primary antibody anti-sarcomeric MyHCs (MF20, DHSB) at room temperature. Cells were washed three times, permeabilized 5 min with 0.25% Triton X-100 in PBS and incubated with secondary FITC anti-mouse antibody (1:500, Sigma-Aldrich) plus DAPI (2 μg/mL) for 40 min at room temperature, light protected. The acquisition was performed by using the inverted microscope Leica DMI6000 B. Three independent immunofluorescence experiments were performed.