Histochemical and immunohistochemical staining

Sections cut from the above blocks were stained with haematoxylin–eosin (H&E) and the collagen stain, Picrosirius Red, as well as being immunostained with antibodies specific for alpha-smooth muscle actin (ASMA), dendritic cell-lysosomal associated membrane protein (DC-LAMP), CD31 (PECAM-1; endothelial cell marker) and Ki67. Immunohistochemical methods for ASMA, DC-LAMP and Ki67 were as previously described⁴². ASMA is expressed by myofibroblasts, cells implicated as the source of extracellular matrix in lung fibrosis, and DC-LAMP is expressed by type II pneumocytes (AEC2), the main source of vascular endothelial growth factor (VEGF) in the lung parenchyma. Cell proliferation is a feature of the tissue response to injury, and Ki67 was used to quantify this aspect of repair.

CD31—antigen retrieval was achieved using pre-warmed 0.05% Pepsin (Sigma P7000) in 10 mM HCl, digestion at 37 °C for 1 h. Non‐specific binding was blocked with 10% Normal Donkey Serum (Sigma D9663) in 1% Bovine Serum Albumin (Sigma A4503)/PBS + 0.05% Tween 20 (10%NDS/BSA/PBS-T). Primary antibodies CD31 (PECAM-1) clone CO.3E-1D4 (Novus Biologicals NB100-65900) and Normal Mouse IgG (Sigma M5284) were diluted to 10 µg/ml in 1:10 blocking solution (10% NDS/BSA/PBS-T diluted in BSA/PBS-T) and incubated overnight at 4 °C. Detection was achieved using biotinylated horse anti mouse (Vector BA2001) and Streptavidin peroxidase polymer (Sigma S‐2438) followed by DAB substrate (Vector SK‐4105) with Haematoxylin counterstain.