Anti cytochrome b5

The lung was inflated in situ via the trachea with PBS containing 3% paraformaldehyde. After 4 h on ice, the tissue was transferred to 50% ethanol. Tissue sections (4 μm) were prepared, deparaffinized with xylene (4 min, x 2), followed by decreasing concentrations of ethanol (100%–50%) and finally, water. After antigen retrieval using citrate buffer (10.2 mM sodium citrate, 0.05% Tween 20, pH 6.0) and quenching of endogenous peroxidase with 3% H₂O₂ for 15 min, sections were incubated for 2 h at room temperature with 10–100% goat serum to block nonspecific binding. This was followed by overnight incubation at 4°C with rabbit IgG or rabbit polyclonal anti-Gal-3 (1:2000; R&D Systems, Minneapolis, MN), anti-iNOS (1:750; Abcam, Cambridge, MA), anti-mannose receptor (MR)-1 (1:1000; Abcam), or anti-cytochrome b5 (1:250; Abcam) antibodies. Sections were then incubated with biotinylated secondary antibody (Vector Labs, Burlingame, CA) for 30 min at room temperature. Binding was visualized using a Peroxidase Substrate Kit DAB (Vector Labs). Random sections from three mice per treatment group were analyzed by light microscopy using an Olympus BX51 microscope (Olympus America Inc., Center Valley, PA).