Fluoviewer software

Confocal images were acquired with an inverted confocal microscope (FluoView FV1000; Olympus, AU), using a 100 X oil immersion objective. A 488-nm helium neon laser was used for excitation of FITC-labelled PuroA and a 561-nm helium neon laser was used for excitation of both PI and SYTO 85 Orange. The imaging sequence cycled among 488 nm fluorescence excitation, 561 nm fluorescence excitation, and differential interference contrast (DIC) with each sequence repeated every 30 s. All images were acquired and analyzed with Olympus FluoViewer software. ImageJ software (version 1.50b) was used for measuring the cell size.

BAL fluid was filtered using a 40 μm cell sieve. BAL cells were pelleted, counted and 1 ×10⁵ viable cells were used to produce cytospin slides (Thermo Shandon Cytospin 3, Thermo Scientific). Cytospin slides were fixed in 4% PFA, washed, and blocked overnight in blocking solution (10% goat serum/1% BSA/2 mM EDTA/HBSS/0.1% Tween-20). Slides were then washed and incubated with anti-histone H2A.X antibody (Abcam, UK) for 1 h before washing. Anti-rabbit Alexa Fluor^® 488-conjugated secondary antibody (Invitrogen, UK) and DAPI (Sigma, UK) were then diluted in blocking buffer for 1 h. Stained slides were then washed, mounted, sealed and visualized using an Olympus inverted fluorescence confocal microscope and analyzed using Fluoviewer software (Olympus).

Lung sections were stained using a modified protocol based on published reports (34 (link), 35 (link)). Five micrometer sections from paraffin-embedded lung biopsies from control or IPF patients were dewaxed prior to heat-induced epitope retrieval with Tris-EDTA buffer, pH 9.0. Sections were blocked with Fc block (BD biosciences, UK) before incubation with a blocking buffer (5% goat serum/2.5% BSA/PBS/0.1% Tween-20) for 1 h. Slides were then washed and incubated with anti-citrullinated histone H3 (Abcam, UK) and anti-MPO (R&D systems, UK) antibodies diluted in 0.5x blocking buffer overnight at 4° C. Anti-rabbit Alexa Fluor^® 647-conjugated and anti-mouse Alexa Fluor^® 555-conjugated secondary antibodies (Invitrogen, UK) and DAPI (Sigma, UK) diluted in 0.5x blocking buffer were then added for 30 min. Stained sections were washed, mounted, sealed and visualized using an Olympus inverted fluorescence confocal microscope and analyzed using Fluoviewer software (Olympus).