Cs sp5 multi photon laser scanning confocal microscope

Cells were seeded on glass coverslips 24 h prior to use for IFA. Infected or uninfected cells were rinsed in cold PBS and fixed with 4% paraformaldehyde (EM Sciences, Hatfield, PA) for 15 min and permeabilized with 0.2% Triton X-100 for 5 min. After washing and blocking in 1% bovine serum albumin (BSA) PBS, the cells were reacted with primary antibodies (1:300, overnight at 4°C) reactive against all structural SeV antigens followed by washing and incubation with fluorescent dye-conjugated secondary antibodies and Alexa 633 donkey anti-goat IgG (Molecular Probes) (1:100; 1 h at 4°C). F-actin was visualized by staining with Alexa 488 phalloidin for 1 h at 37°C according to the manufacturer’s instructions (Molecular Probes). Cells were mounted in Vectashield with DAPI to stain the nucleus (Vector Laboratories, Burlingame, CA). Fluorescence and confocal microscopy assessments were performed with a Leica CS SP5 multiphoton laser scanning confocal microscope (Leica Microsystems, Weitzler, Germany) and quantitated using ImageJ software (National Institutes of Health). Images were processed using Adobe Photoshop CS4 (Adobe, San Jose, CA). More than 10 cells (from two coverslips) were analyzed for each condition.

DU145 cells were grown on glass coverslips and mock treated or treated with CpG-ODN (1 µM) or TNFα (10 ng/mL; R&D Systems, Inc., Minneapolis, MN, USA) for 1 hour with or without pretreatment with EGCG for 24 hours. Cells were rinsed with phosphate-buffered saline (PBS) and permeabilized with 0.1% Triton-X100 for 15 minutes. After 1 hour of incubation with NF-κB p65 subunit antibody (1:250), cells were washed once with 0.01% Tween 20 followed by two washes with PBS. Cells were incubated for 1 hour with Alexa Fluor 488 donkey anti-rabbit secondary antibody (1:400). Cells were washed with 0.01% Tween 20 and PBS, mounted in VECTASHIELD^® with 4′,6-diamidino-2-phenylindole (DAPI; Vector Labs, Burlingame, CA, USA). Cells were imaged with a Leica CS SP5 multi-photon laser scanning confocal microscope (Leica Microsystems, Weitzler, Germany).