Quantifying Tumor Cell Proliferation

Ki-67 immunostaining was conducted on tumors from treprostinil treated and control animals as as previously described(22 (link), 23 (link)). In brief, 5 micron lung sections from the urethane, ET, and LT groups were deparaffinized, peroxidases blocked with 3% hydrogen peroxide, and antigen retrieval performed in boiling Diva Decloaker (Biocare Medical, DV2004G1) reagent under pressure for 10 min. Sections were incubated with Ki67 primary Ab (1:25 dilution, Abcam 15580) for 30 min. at room temperature, and Ki67⁺ nuclei were detected using Biocare Medical’s Mach 2 detection kit (MRCT523). Ki67 positive nuclei/mm² were counted in each tumor and averaged within each group using an Olympus BX51 microscope fitted with an Olympus DP72 digital camera and CellSens Entry software (Olympus) to calculate tumor area.