Rabbit anti prosp c

Mouse lungs were perfused with 10 mL of saline (through the right ventricle), harvested, and fixed overnight with 4% paraformaldehyde. Fixed samples were embedded in Tissue-Tek® O.C.T. compound (SAKURA Finetek) and serially sectioned (3 μm thickness). The tissues were incubated in 0.3% H₂O₂/methanol for 30 min to block endogenous peroxidase activity. The sections used for CD31 staining were heat-treated in 0.01 M citrate buffer (pH 6.0) for antigen retrieval before peroxidase blocking. Tissues were blocked with 5% goat serum and incubated with appropriate primary antibodies overnight at 4 °C, followed by secondary antibodies for 60 min at room temperature. The following primary antibodies were used: rabbit anti-mouse BLT2 (5 μg/mL; generated in our laboratory by immunizing a rabbit with a BLT2 C-terminal peptide); rabbit anti-proSP-C (1:1000; Millipore); hamster anti-T1α (1:2000; BioLegend); rabbit anti-CD31 (1:50; abcam), rabbit IgG (5 μg/mL; DAKO); and hamster IgG (1:2000; BioLegend). The secondary antibodies were biotinylated anti-rabbit goat IgG (1:300; DAKO) and biotinylated anti-hamster goat IgG (1:200; Vector Laboratories). Tissues were labeled with horseradish peroxidase-conjugated streptavidin (DAKO), and the signals were detected by incubating with diaminobenzidine. Images were captured under a microscope (Keyence BZ9000).