Alex fluor 594 goat anti rabbit igg h l

After the stimulation assay, epithelial and stromal cells were washed three times in sterile PBS, fixed for 10 min in 4% paraformaldehyde (Sigma), washed two times in PBS- 0.1% Triton for 5 min and incubated with the blocking solution (PBS + 2.5% BSA) for 1 h at room temperature. Cells were incubated overnight at 4 °C with rabbit polyclonal anti-IRF3 antibody (1:50, ab25950, Abcam^®, Cambridge, UK) and rabbit polyclonal anti-NFkB p65 antibody (1:100, sc-109-G, Santa Cruz biotechnology^®, Texas, USA). After washing twice for 10 min in PBS, the secondary antibody (1:300, Alex Fluor^® 594 Goat Anti-Rabbit IgG (H + L), Life Technologies, Carlsbad, USA) was added and cells incubated for 1 h at room temperature. After washing for 10 min in PBS, the glass coverslips were mounted in Vectashield^® mounting medium with DAPI (H-1200, Vector Laboratories Inc., Burlingame, CA, USA). Slides were observed using a fluorescence microscope (Leica DM5000B) and the images obtained in Adobe Photoshop CS5.

The reference compounds of calycosin, formononetin, astragaloside IV (AS-IV), ferulic acid and ligustilide were purchased from Chinese National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). Avertin and simvastatin were purchased from Sigma-Aldrich (St. Louis, MO). The anti-CD45, anti-CD31, α-tubulin, VEGF, p-eNOS, eNOS and biotin-conjugated goat anti-rabbit IgG polyclonal antibodies were obtained from Abcam (Cambridge, UK). Caspase-3, cleaved caspase-9, Bcl-2, Bax, Bad, p-Akt, Akt and GAPDH antibodies were bought from Cell Signalling Technology (Boston, MA). The in situ cell death detection kit and protease and phosphatase inhibitor cocktails were purchased from Roche Diagnostics (Mannheim, Germany). Interleukin 6 (IL-6) and interleukin 1 beta (IL-1β) Mouse ELISA kits were obtained from R&D Systems (Minneapolis, MN). Alex Fluor 594 Goat Anti-Rabbit IgG (H + L) was from Life Technologies (Waltham, MA).