Dapi p36935

HFL1 cells were grown on coverslips and treated with or without 100 ng/ml His‐PTX3 fusion protein for 24 h. Cells were fixed in 4% paraformaldehyde for 20 min and incubated with antibodies against His (sc‐803, Santa Cruz) and CD44 (GTX83114, GeneTex). For lung sections, samples were incubated with PTX3 (1:100, ab90806; Abcam), fibronectin (1:350 dilution, #15613‐1‐AP, ProteinTech) and α‐SMA (1:100 dilution, GTX100904, GeneTex) at room temperature for 1 h. Samples were incubated with Alexa 568‐ or 488‐conjugated secondary antibodies (Invitrogen) at a 1:200 dilution. The samples were washed with 0.1% Tween 20 in PBS and stained with DAPI (P36935, Invitrogen) on coverslips. The fluorophores were excited by a laser at 405, 488, or 543 nm and observed using an FV‐3000 confocal system (Olympus). Randomly chosen fields were photographed at ×200 magnification under a fluorescence microscope.

Cells were fixed using 2% formaldehyde in phosphate-buffered saline (PBS) for 15 minutes at room temperature and were then permeabilized by incubating for 30 minutes in ethanol 70% at −20°C. After washing 3 times in PBS-0.02%Tween and blocking in PBS-5% serum during 15 min, the cells were incubated for 4 hour at room temperature with an anti-PML antibody (mouse monoclonal IgG₁, Santa Cruz : sc-966, (1/50)) diluted in the blocking buffer. After washing 3 times in PBS-0.02% tween and blocking in PBS-5% serum during 15 min, cells were incubated with the secondary antibody 1/200 diluted (Goat anti-Mouse IgG secondary antibody Alexa 488, Invitrogen-Molecular Probes : A-10680) for 1 hour at room temperature in the dark. Cells were then washed 3 times in PBS-0.02%Tween, and covered with Anti-Fading reagent with DAPI (Invitrogen Prolong R. Gold antifade reagent with DAPI P36935). Slides were then analyzed by confocal microscopy.