Bx61 motorized microscope

HUVEC monolayers in 24-well plates were uninfected or infected with PR8 or CA07 at a MOI of 20~40. At 12 or 24 hour after infection, the virus was removed from the HMVECs. The cells were washed with phosphate-buffered saline (PBS) for three times and then fixed in 30% ice-cold acetone for 1 hour at 4°C. Viral nucleoprotein (NP) protein was detected by using mouse monoclonal antibody (1:1000, 0.3 ml) for 60 minutes at 37°C(Chemicon International, Inc.). The secondary goat anti-mouse antibody labeled with fluorescence was used for 60 minutes at 37°C. After adding 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, 1:10, 0.3 ml) and incubating for 10 minutes, the cells were washed with PBS for three times. Fluorescent images were acquired by MicroSuite FIVE software (Olympus Soft Imaging Solutions) with an Olympus BX61 motorized microscope (Olympus). The infection rate of HUVECs was calculated as the percentage of infected cells divided by the total cells.

Cells were grown in wells of 4-chamber culture collagen-coated slides. Cells were fixed in 4% paraformaldehyde for 15 min at 4°C, washed with PBS, and permeabilized for 5 min with 0.1% Triton-X-100. After blocking with PBS + 2% BSA + 0.1% Tween-20, cells were incubated with primary antibody against β-catenin and goat anti-rabbit IgG for 45 min each. Images were acquired by MicroSuite FIVE software (Olympus Soft Imaging Solutions, Golden, CO, USA) with an Olympus BX61 motorized microscope (Olympus America, Center Valley, PA, USA).

Blastomeres isolated at the 64-cell stage were cultured in agar-coated dishes, and successive cell divisions were recorded with a BX61 motorized microscope (Olympus) equipped with a water-immersion lens and digital camera. The DIC images were acquired using LuminaVision software (Mitani Co., Tokyo, Japan) at one frame every 3 minutes for approximately 11 hours.