Goat anti mouse alexa fluor 488 and

hESC-OPs^GFP+ cells differentiated under osteogenic conditions for 14 days were washed with PBS and fixed in 4% paraformaldehyde for 20 minutes at room temperature. For detection of intracellular proteins, the cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS and blocked with 10% goat serum/2% BSA in PBS for 1 hour at room temperature. Then the cells were incubated with primary antibodies against mouse anti-human GFP (1 : 200, Santa Cruz Biotech), rabbit anti-human collagen-1 (1 : 300, Millipore), rabbit anti-human osteocalcin (1 : 200, AbD Serotec), and rabbit anti-human alkaline phosphatase (1 : 200, Santa Cruz Biotech) in blocking solution at 4°C overnight. Then, the cultures were washed thrice in PBST (0.1% Tween-20 in PBS) and incubated with goat anti-mouse Alexa Fluor-488 and goat anti-rabbit Alexa Flour-594 conjugated secondary antibodies (1 : 200, Molecular Probes) for 1 hour at room temperature, followed by nuclear labelling with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) for 5 minutes at room temperature. The cultures were then washed thrice with PBS. Immunostained cultures were observed and imaged using Olympus IX70 fluorescence microscope.

Trachea were excised from newborn piglets and immediately fixed in 4% paraformaldehyde (EMS) in PBS for 1 hr at room temperature. Tissues were then placed in 30% sucrose and incubated overnight at 4°C, followed by quick-freezing in OCT using a dry ice/EtOH bath and stored at −80°C. Prior to immunocytochemistry, frozen blocks of tissue were cryosectioned at 7 μm followed by permeabilization in 0.3% TX-100 (Thermo-Fisher) in PBS for 20 min, and blocked in Super-Block (Thermo-Fisher) with 5% normal goat serum (Jackson ImmunoResearch) for 1 hr, all at room temperature. Tissue sections were then incubated for 2 hr at 37°C with indicated antibodies: β-tubulin IV(1:300, Biogenex), MUC5AC (1:5000, Novus Biologicals), MUC5B (1:2000, Santa Cruz). Sections were then incubated for 1 hr with secondary antibodies goat-anti-mouse Alexa-Fluor-488 and goat anti-rabbit Alexa-Fluor-555 (1:1000, Molecular Probes/Invitrogen) and phalloidin-633 (1:300, Molecular Probes/Invitrogen). Slides were imaged on an Olympus Fluoview FV3000 confocal microscope with a Plan.ApoN 60X oil lens. Images were post-processed using the Olympus imaging software, CellSens.