Lysates of normal lung fibroblasts (WI38) and of human fibrosarcoma (8387) were probed with the PTX3, pAb, over night at +4 °C at a concentration of 500ng/ml in 5% dry milk in PBS+/+ containing 0.05% Tween 20. The signal was revealed with an HRP-conjugated anti-rat IgG secondary antibody in 5% dry milk PBS+/+ containing 0.05% Tween 20 after incubation for 1 hour at room temperature and IMMOBILON Western Chemiluminescent substrate

Pulmonary PTX3 expression by immunohistochemistry (IHC) using PTX3, pAb. Method: Photomicrograph of PTX3 stained lung tissue of Cigarette Smoke (CS)-exposed WT mice. Mice were exposed to CS for 4 weeks (subacute).(V: vein, A: artery, AW: airway. Magnification 200×). In courtesy of Guy Brusselle.

Overlay histogram showing HUVEC cells stained with PLIN3 (green line). The cells were fixed with 2% paraformaldehyde (10 min) and then permeabilized with 90% methanol for 10 min. The cells were then icubated in 2% bovine serum albumin to block non-specific protein-protein interactions followed by the antibody (PLIN3, 1:25 dilution) for 60 min at 37ºC. The secondary antibody used was Goat-Anti-Mouse IgG, DyLight® 488 Conjugated Highly Cross-Adsorbed at 1/200 dilution for 40 min at 37ºC. Isotype control antibody (blue line) was mouse IgG1 (1ug/1x10^6 cells) used under the same conditions. Acquisition of >10,000 events was performed.