Methyl green

Mice were killed by carbon dioxide (CO₂) inhalation and perfusion fixed with 10% buffered formalin via the left ventricle for 5 minutes. Then, the ankles with Achilles tendons were dissected and fixed in 4% paraformaldehyde for 24 hours. All of these specimens were decalcified in a 10% EDTA solution for 1 month, embedded in paraffin and cut into 5‐μm sections for staining.
Trap staining was performed following the manufacturer's protocol (Sigma‐Aldrich, 387A‐1KT), followed by counterstaining with Methyl Green (Sigma‐Aldrich, M884).
Sections were stained with 0.1% Safranin O and 0.02% Fast Green (Sigma‐Aldrich) according to the manufacturer's instructions.
Immunohistochemical staining was carried out with primary antibodies against IL‐17, IL‐17R (Abcam, Cat No. ab11370) and β‐catenin (Cell Signaling Technology, Cat No. 4370) with a 1:1000 dilution of an appropriate secondary antibody. Protein expression was visualized with a DakoCytomation EnVision staining kit. The mean density of the positive area was measured by Image‐Pro Plus 6.0 (IPP) image analysis software. Three random slides were selected, and five random fields of images per sample were taken.

On the day of graft harvest, the anesthetization took place 10 minutes after the intravenous administration in the tail vein of dextran‐biotin (Sigma, 14402; 80 mg/kg in 50 μL). Four‐μm tracheal sections (n = 6 per group) from dextran‐biotin‐treated animals were incubated directly with streptavidin peroxidase (Sigma, E2886, 1:500). Vessels were revealed with 3,3′‐diaminobenzidine, and nuclei were stained in a solution of Methyl Green (Sigma, 198080, 5 g/L, pH 4.2).

Paraffin-embedded 4 µm tissue sections were stained with haematoxylin and eosin (H&E) and analyzed for inflammation and tissue damage as described [22] (link)–[25] (link). Briefly, all slides were coded and scored by a pathologist blinded for the experimental groups. Lung tissues were scored for the following parameters: interstitial inflammation, necrosis, endothelialitis, bronchitis, edema, pleuritis, presence of thrombi and percentage of lung surface with pneumonia. All parameters were rated separately from 0 (condition absent) to 4 (most severe condition). The total histopathological score was expressed as the sum of the scores of the individual parameters, with a maximum of 24. Granulocyte stainings, using fluorescein isothiocyanate-labeled rat-anti-mouse Ly-6G mAb (BD Pharmingen, San Diego, CA) were done as described previously [23] (link)–[25] (link). Slides were counterstained with methylgreen (Sigma-Aldrich, St. Louis, MO). The total tissue area of the Ly-6G-stained slides was scanned with a slide scanner (Olympus dotSlide, Tokyo, Japan) and the obtained scans were exported in TIFF format for digital image analysis. The digital images were analyzed with ImageJ (version 2006.02.01, National Institutes of Health, Bethesda, MD) and the immunopositive (Ly6G+) area was expressed as the percentage of the total lung surface area.

Polarized CFBE cells were fixed with PFA 4% (vol/vol), permeabilized with Triton X-100 (17-1315-01; Amersham Biosciences) 0.5% (vol/vol), and blocked with BSA 1% (wt/vol) before being removed from their supports using a scalpel. Cells were then incubated overnight at 4°C with primary antibodies, after which a mix of the secondary antibodies and nuclear dye (4 μg/ml, Methyl Green, 67060; Sigma-Aldrich) was applied for 2 h at RT. Negative controls were performed for each experiment by adding BSA instead of primary antibodies (Fig S7). Filter sections were mounted in a mix of N-propyl gallate (P3130; Sigma-Aldrich) and Glycerol for microscopy (104095; Merck). A list of primary antibodies can be found in Table S1.
Imaging was performed with a Leica TCS SP8 confocal microscope, using HC Plan Apo 20x/0.75 and HC Plan Apo 63x/1.4 objectives. Software used for acquisition was Leica's LAS x, and image processing was performed on ImageJ FIJI (81 (link)). FIJI was used to generate average image projections.

Tissue sections were dewaxed and rehydrated through a descending alcohol gradient, then immersed in boiled 10 mM sodium citrate, 0.005% Tween 20, pH 6.0 for 20 min to expose antigen sites. Endogenous peroxidase activity was quenched with H2O2 before staining with primary antibody (diluted in PBS with 0.1% Triton-X 100 and 3% goat serum) overnight at 4 °C in a humidified chamber, then biotinylated secondary antibody was applied (Vector Laboratories, CA, USA) for 2 h at room temperature (RT), and streptavidin-conjugated-Horseradish Peroxidase (Sigma, MO, USA) was added for 1 h. Chromogenic detection was with 3,3-diaminobenzidine (DAB) in the presence of H2O2 for 5 min, slides were counterstained for 2 min in Methyl Green (Sigma, MO, USA), washed in H₂O or water dehydrated and mounted in DPX mounting medium. The images were viewed using an Olympus Slide Scanner.