Quantifying Pulmonary Cell Proliferation

Cell proliferation in the rats was analysed by quantifying the pulmonary cells that incorporated the exogenously supplied 5-ethynyl-2′-deoxyuridine (EdU) to identify the cells undergoing DNA replication. EdU was intraperitoneally injected into the rats at 50 μg/g 24 h before sacrifice [21 (link)]. EdU staining was performed using the Click-iT EdU imaging kit (C10337, Life Technologies) on frozen sections according to the manufacturer’s instructions. To characterize the EdU-positive cells, we performed double-immunostaining with FITC-labelled anti-α-SMA (clone 1A4, Sigma-Aldrich, Lyon, France, 1/100) or with anti-CD34 (EP373Y, Abcam, Amsterdam, 1/200). Images were captured with an LSM 700 confocal microscope (Carl Zeiss, Le Pecq, France). Images were recorded and analysed with ZEN software (Carl Zeiss).