NETs are formed in the lungs of mice with TRALI. (A) Lung tissue cryosections from control healthy mice (control) and from mice with TRALI (2 hours after antibody injection, TRALI) were immunostained for Gr-1 (orange) and stained for DNA (Hoechst 33342, blue) and then observed by widefield fluorescence microscopy. White arrowheads point at NET structures probably originating from Gr-1-positive cells. a indicates alveolus. Scale bar represents 20 μm. (B) Multiphoton fluorescence micrographs of a lung tissue cryosection from an LPS-treated mouse (LPS + Ctrl Ab) and from a mouse with TRALI (LPS + H-2K^d Ab). The sections were immunostained for VWF (red) to show blood vessels, histone H3Cit (green), and counterstained for DNA (Hoechst 33342, blue). Three independent experiments were immunostained. Scale bars represent 200 μm. (C) Multiphoton fluorescence micrographs of lung tissue cryosections from a mouse with TRALI. Five representative sequential sections 2.5 μm apart are shown. Tissue cryosections were stained as in panel B. v indicates vessel. Scale bars represent 10 μm. The z-stack and the 3-dimensional reconstruction of the specimen are online as supplemental Videos 1 and 2.