Anti mouse igg dylight 594

CMs derived from IMR hiPSCs were fixed with 4% paraformaldehyde and were permeabilized in PBS plus 0.5% Triton X-100 for 0.5 h. After blocking in a 5% normal goat serum, 5% normal donkey serum, 3% BSA, and 0.1% Tween 20 in PBS for 16 h at 4°C, the cells were incubated with an α-actinin (mouse monoclonal IgG, 1 : 1000; Sigma), troponin T2 (TnT2, mouse monoclonal IgG, 1 : 200, Santa Cruz Biotechnology, Dallas, TX, USA), MLC2v (rabbit polyclonal IgG, 1 : 200; Proteintech Group, Rosemont, IL, USA), or β-MHC (mouse MYH7 monoclonal IgM, 1 : 100; Santa Cruz Biotechnology) antibody for 16 h at 4°C. The cells were then washed and incubated with different secondary antibodies diluted in blocking buffer (1 : 1000): DyLight-594 anti-mouse IgG, DyLight 488 anti-mouse IgG, DyLight-594 anti-rabbit IgG, or DyLight-488 anti-mouse IgM (all from Life Technologies) at 25°C for 1 h. Nuclei were visualized by DAPI (Wako).

Cells that were grown on chambered cover slips were infected with HCMV strain NR-1 at a multiplicity of infection (MOI) of 5. At 6 h posttransfection, cells were fixed with 4% formaldehyde and blocked with 4% bovine serum albumin (BSA) in PBS and stained with primary mouse IE1/2 antibody (ab53495, Abcam, Cambridge, UK), and then incubated with the secondary antibody Dylight 594 anti-mouse IgG (Life Technology). Cell nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Images were captured with a Nikon Eclipse TE300 microscope (Diagnostic Instruments, Inc., Sterling Heights, MI, USA) [20 (link)]. The digital images were subsequently merged using FV10 ASW V4.1 software (Olympus, Tokyo, Japan).