Shandon varistain 24 4

Lung tissue was fixed in 4% paraformaldehyde and embedded in paraffin using a Leica ASP 300 tissue processor (Leica, Bannockburn, IL). Microtome sections were cut at 5-μm thickness and stained with hematoxylin and eosin (H&E) using a Shandon Varistain 24–4 (Thermo Fisher Scientific). Alternatively, sections were stained using periodic acid-Schiff (PAS) reagent. The level of peribronchial inflammation (H&E stained) or mucus production (PAS stained) was analyzed by microscopy and the transmitted light images were collected on a Nikon Eclipse 800 microscope equipped with an Olympus DP 71 camera and cellSens software (Version 1.9).

Lung tissue was fixed in 4% paraformaldehyde and embedded in paraffin using a Leica ASP 300 tissue processor (Leica, Bannockburn, IL, United States). Microtome sections were cut at 5 μm thickness and stained with hematoxylin and eosin (H&E) using a Shandon Varistain 24-4 (Thermo Fisher Scientific). In addition, sections were stained using Gomori’s Trichrome (EMD Chemicals, Gibbstown, NJ, United States) for histological analysis using a Thermo Shandon automated stainer. The level of pulmonary or peribronchial inflammation (H&E stain) and collagen deposition (Trichrome stain) was analyzed by microscopy and the transmitted light images were collected on a Nikon Eclipse 800 microscope equipped with an Olympus DP 26 camera and cellSens software (Version 1.9).

Explanted samples (Figure 2C) were washed in PBS and fixed overnight with 4% paraformaldehyde at 4°C. Fixed tissues were processed in a Thermo Excelsior AS (Thermo Fisher Scientific, Waltham, Massachusetts, United States) and embedded with a Thermo HistoStar (Thermo Fisher Scientific) machine. Five-micrometer-thick sections were cut using a Shandon Finesse 325 microtome (Thermo Fisher Scientific). Slides were stored at 37°C overnight to dry completely before staining. Hematoxylin and eosin, and Van Gieson’s stainings were performed using a Shandon Varistain 24–4 (Thermo Fisher Scientific) automated machine. Von Kossa staining was carried out using a Silver plating kit (In Vitro Diagnostic Medical Device, Darmstadt, Germany) for the detection of microcalcification. Collagen and elastin contents of the explants in the Van Gieson’s staining were quantified using ImageJ software via color deconvolution, to separate and convert the collagen and elastic fibers to the mean Gray value of the pink and purple and colour intensity, respectively. Threshold values were set for each channel and area-based analysis was used to extract and quantify the regions of interest (ROIs) from the image. Results were expressed as proportion of area occupied by collagen or elastin within the graft tissue.