Immunohistochemistry on Fixed Lung Tissue

Excised lungs were fixed with 4% formaldehyde for 48h, and embedded in paraffin for slide preparation. Before staining, 5-μm slides were dewaxed by xylene and rehydrated by a graded series of ethanol. The sections were stained by hematoxylin and eosin (HE) to evaluate pathogenesis. The procedure of IHC was described in the previous study [40 (link), 41 (link)]. Ten % BSA-blocked slides were incubated with the primary antibody targeting IL10 (Bioss Inc., Woburn, MA, USA), CD163 (abcam^®, Cambridge, MA, USA), EGFR (Cell Signaling Technology, Beverly, MA, USA) and p-Src (Y419; GeneTex, Inc., Irvine, CA, USA) for 1h, at room temperature. Subsequent procedures were performed using Vectastain ABC kit (Vector Laboratories, Burlingame, CA) according to the instruction, and photographed by photomicroscope (Olympus, Melville, NY, USA).