AT inhibits HRPII-mediated activation of neutrophils in vivo. Mice were injected IP with saline or HRPII (10 µg/g) and AT (500 µg/mouse ∼20 µg/g) followed by collection of organs after 3.5 hours for analysis. (A) Cryosections of the lung tissue were fixed and permeabilized followed by analysis of infiltration of neutrophils to the lung with anti-MPO (rabbit) antibody and Alexa Fluor 555-conjugated anti-rabbit antibody. DAPI was used to stain the nucleus. Relative intensity of the MPO stain is presented. (B) Mac1 surface expression in Ly6G-positive mouse neutrophils was analyzed by flow cytometry as described in "Materials and methods." (C) The MPO level in plasma (marker of neutrophil activation) was analyzed by the MPO activity assay as described in "Materials and methods." (D) The same as (A) except that the lung cryosections were fixed, permeabilized, and incubated with anti-citrullinated histone H3 (rabbit) and anti-Ly6G (rat) antibodies followed by Alexa Fluor 555-conjugated anti-rabbit and Alexa Fluor 488-conjugated anti-rat antibodies. DAPI was used to stain the nucleus. The arrows indicate citrullinated histone H3. (E) The PBS-perfused lower right lobe of lung was dissected and dissolved in the tissue lysis buffer followed by western blotting of the lysate for detection of MPO, citrullinated histone H3, histone H3, and β-actin using appropriate antibodies. Densitometric analysis of expression of these proteins is presented.