Immunofluorescence microscopy. HMVEC-d or HUVEC cells grown in eight-chamber slides were infected or left uninfected for different times and stained for primary selleck chemical Z-VAD-FMK antibody to EEA-1, LAMP 1, phalloidin-Alexa Fluor 488, Rab5, and Rab34 antibodies, as well as antibodies against viral glycoprotein gpK8.1A and ORF65 capsid protein, wherever applicable and then stained with anti-mouse or anti-rabbit antibodies against endocytic markers and viral markers. For transferrin and dextran colocalization with virus, cells were incubated with Texas Red-labeled dextran (0.5 mg/ml) and KSHV at an MOI of 10 or with 35 ��g/ml of Alexa Fluor 594-conjugated transferrin and KSHV at an MOI of 10 for 5, 10, and 15 min at 37��C. The cells were washed in HBSS, fixed in 2% paraformaldehyde for 15 min, permeabilized with 0.

2% Triton X-100 for 5 min, and blocked with 5% bovine serum albumin (BSA) for 15 min. Cells were incubated with anti-gpK8.1A mAb at room temperature for 1 h and stained with Alexa Fluor 488-conjugated goat anti-mouse secondary antibodies at room temperature for 1 h. For colocalization studies with virus and caveolin, cells were incubated with KSHV at an MOI of 10 for 5, 10, and 15 min at 37��C, followed by fixation with 4% paraformaldehyde for 15 min, and permeabilized with 0.2% Triton X-100 for 5 min at room temperature. After being blocked for 15 min with 10% BSA, cells were processed for double immunostaining with antibodies against gpK8.1A and caveolin. This was followed by 1 h of incubation at room temperature with goat anti-mouse antibody labeled with Alexa Fluor 488 and goat anti-rabbit antibody labeled with Alexa Fluor 594.

Following washing with phosphate-buffered saline (PBS), slides were mounted with mounting medium containing 4��,6��-diamidino-2-phenylindole (DAPI) and examined under a Nikon fluorescent microscope equipped with a Metamorph digital imaging system. Laser-scanning confocal immunofluorescence microscopy. HMVEC-d cells infected with KSHV at an MOI of 10 were fixed with 4% paraformaldehyde. Cells were then permeabilized with 0.2% Triton X-100 for 5 min, washed, and blocked with goat serum for 30 min. The samples were then incubated for 1 h with primary antibody to gpK8.1A, Rab5, or Rab34 for 1 h at room temperature. The cells were then stained with appropriate secondary antibody linked with Alexa Fluor 488 or Alexa Fluor 594.

An Olympus Fluoview 300 fluorescent confocal microscope was used for imaging, and analysis was performed using Fluoview software (Olympus, Melville, NY). Electron microscopy. HMVEC-d cells grown in 75-cm2 flasks were left untreated and washed three or four times with PBS. The cells were infected with KSHV Drug_discovery at an MOI of 20 at 4��C for 1 h, and then the temperature was shifted to 37��C and infection was done for 5 and 10 min. After each time point, cells were washed and pelleted.