Af488 anti rabbit secondary antibody

HEPES (4-(2-hydroxyethyl)–1-piperazineethanesulfonic acid), PEI 25 kDa, heparin sodium salt, Paraformaldehyde solution, FluorSave™ Reagent, Eagle’s Minimum Essential Medium (EMEM), RPMI-1640 Medium, fetal bovine serum (FBS), Penicillin-Streptomycin solution, Dulbecco’s Phosphate Buffered Saline (PBS), trypsin-EDTA solution, 200 mM L-glutamine solution, Paraformaldehyde, Tween20, agarose and Alcian Blue solution (1% in 3% acetic acid pH 2.5) were purchased from Sigma-Aldrich (Darmstadt, Germany). Lipofectamine 2000, SYBR gold dye, AF488-anti-rabbit secondary antibody, rhodamine phalloidin, 4′,6–diamidino–2-phenylindole dihydrochloride (DAPI), Alexa Fluor™ 647 NHS ester and Alexa Fluor™ 488 NHS ester were obtained from Life technologies (Carlsbad, California, USA). Transwell® polyester membrane cell culture inserts (0.4 μm pore size) were purchased from Corning (New York, USA). PneumaCult ALI differentiation medium, hydrocortisone and heparin were purchased from Stemcell Technologies (Vancouver, Canada). Alveofact was purchased from Lyomark Pharma (Oberhaching, Germany). ROTI®GelStain Red and bovine serum albumin were purchased from Carl Roth GmbH (Karlsruhe, Germany). Dicer substrate double-stranded siRNA (DsiRNA) targeting human GAPDH, non-specific DsiRNA and amine-modified siRNA were purchased from integrated DNA Technologies (Leuven, Belgium).

Cells for immunofluorescent staining were grown and treated in chamber slides, and then fixed in 4% formaldehyde in PBS for 10 minutes, permeabilized for 10 minutes with 0.2% Triton X-100 in PBS, and blocked with 2% BSA for 1 hour. Rabbit primary antibody to p65 (Cell Signaling®) was diluted at 1:400 in PBS containing 1% BSA and incubated for 1 hour at room temperature. AF-488 anti-rabbit secondary antibody was from Life Technologies® (Grand Island, NY), and was diluted 1:250 in 1% BSA in PBS, and incubated for 1 hour. Images were captured using Olympus® BX53 optical microscope.