Dotslide scanner

Paraffin-embedded lung was cut into 4-μm sections and stained with hematoxylin and eosin (H&E). Slides were coded and scored by a pathologist blinded for group identity as described [19 (link)]. To determine neutrophil influx in the lung, sections were stained with anti-mouse Ly6G mAb (BD Biosciences) as previously described [29 (link)]. Slides were scanned using an Olympus dotSlide scanner (Olympus, Tokyo, Japan) to generate TIFF images of the full tissue section. Ly-6G positivity was measured using ImageJ; the amount of positivity was expressed as percentage of the total lung surface area [29 (link)].

Stained sections were photographed with an upright AX10 microscope (Zeiss) linked to a Retiga 400R camera (QImaging) using a QImagin pluggin for ImageJ/FiJi. Images were processed and assembled using Adobe Photoshop. Some sections were photographed using a Dotslide scanner (Olympus) and images were analysed using the OlyVia software (Olympus).

Four‐micrometer tissue sections were stained with hematoxylin and eosin (H&E) and scored as previously described 24 with respect to the following parameters: bronchitis, edema, interstitial inflammation, intra‐alveolar inflammation, pleuritis, endothelialitis, and percentage of the lung surface demonstrating confluent inflammatory infiltrate. Each parameter was graded 0–4, with 0 being ‘absent’ and 4 being ‘severe’; the total pathology score was expressed as the sum of the score for all parameters. Granulocyte staining used FITC‐labeled rat anti‐mouse Ly‐6 mAb (clone 1A8; BD Pharmingen, San Diego, CA, USA; catalog number 551460; 1:20 000). The amount of Ly‐6G positivity was expressed as a percentage of the total surface area. Fibrinogen staining on lung sections was performed as described previously 25 using a biotin‐labeled goat anti‐mouse fibrinogen antibody (Accurate Chemical & Scientific, Westbury, NY, USA; catalog number YNGMFBGBio; 1:500). Slides were scanned using an Olympus dotSlide scanner (Olympus, Tokyo, Japan) to generate TIFF images of the full tissue section. Ly‐6G and fibrinogen positivity was measured using ImageJ; the amount of positivity was expressed as a percentage of the total lung surface area 21, 25.