Ni e brightfield microscope

Immunohistochemistry was performed on 4-μm-thick sections of paraffin-embedded specimens using anti-Ki67 (ab15580, abcam) at 4 degrees in a moist chamber overnight. Then treated with the biotin-labelled secondary antibody, a VECTASTAIN Elite ABC peroxidase kit (PL-6100, Vector laboratories), followed by colour development with DAB. 40× images were acquired with Ni-E brightfield microscope (Nikon, Tokyo, Japan)

Lung sections were immunostained for CD45-expressing cells using rabbit anti-mouse CD45 antibody (Cell Signaling #70257) at 1:1000 concentration using ABC Vectastain kit (Vector Laboratories PK-4000) per manufacturer protocol. Lung lobes were tile scanned at 4X magnification and high resolution on a NiE brightfield microscope (Nikon). Using Nikon elements analysis software, a binary object for lung tissue was defined as saturation above threshold and CD45-positive cells defined as intensity below threshold with a size limit of 5 to 20 square microns. The same analysis was applied to all images, and the number of CD45-positive cells per mm² of lung tissue was calculated.