Intravital Microscopy of Mouse Lung

Mice were anesthetized with Ketamine (80 mg/kg) and Xylaxine (12 mg/kg) i.p. and placed on a custom, heated microscope stage. PE-90 tubing was inserted into the trachea and sutured into place to facilitate mechanical ventilation with a rodent ventilator (Kent Scientific). Mice were ventilated with pressure control ventilation (12–15 cmH₂O), a respiratory rate of 115 breaths per minute, FiO2 of 0.5–1.0, and PEEP of 3 cmH₂O. Isoflurane was continuously delivered at 1% to maintain anesthesia and mice were given an i.p. bolus of PBS (1 ml) prior to the thoracic surgical procedure. The mice were then placed in the right lateral decubitus position and three left anterior ribs were resected and the left lung was carefully exposed. The thoracic suction window attached to a micromanipulator on the microscope stage was then placed into position and 20–25 mmHg of suction was applied (Amvex Corporation) to gently immobilize the lung. The two-photon microscope objective was then lowered into place over the thoracic suction window and a 12 mm coverslip. For intravenous injections, the right jugular vein was cannulated with a 30 gauge needle attached to PE-10 tubing for injections of cells or intravascular dyes.