Intravital Lung Microscopy Protocol

Intravital microscopy of the lung was performed as described previously8 (link)66 (link). Briefly, animals were anaesthetized and mechanically ventilated. A thoracotomy was performed to expose the right middle lung lobe and the lung was hold in position using a custom-built fixation device with an integrated observation window66 (link). Immediately before imaging animals were injected with an Alexa488-coupled anti-Gr1 antibody (clone RB6-8C5, 5 μg per mouse, purified from hybridoma supernatant) and a PE-coupled anti-CD41 antibody (clone MWReg30, 5 μl per mouse, BD Biosciences, Franklin Lakes, NJ, USA). High-speed multichannel fluorescence microscopy was performed on an upright microscope (Axioskop; Carl Zeiss, Göttingen, Germany) equipped with a Lambda DG-4 ultra high speed wavelength switcher (Sutter Instruments, Novato, CA, USA) and a 40 × 0.75 NA saline immersion objective. Videos were recorded with a digital camera (Sensicam QE) and analysed with Slidebook Software (Version 5; Intelligent Imaging Innovations, Göttingen, Germany).