Rabbit anti psmad3

We performed immunohistochemistry (IHC) staining as previously described.^{15 (link)} Primary antibodies used were rabbit anti-Ki67 (1:500) (Cell Signaling Technology, 12202), rabbit anti-NFκB-p105/p50 [p Ser337] (1:300) (Novus Biologicals, NB100–82074), rabbit anti-pSmad3 (1:400, Abcam, ab52903), rabbit anti-pH2AX (1:100) (Cell Signaling Technology, 9718), rabbit anti-myeloperoxidase (Ready-to-Use, Dako, GA51161–2), rabbit anti-hemagglutinin (HA) (1:700) (Cell Signaling Technology, 3724), rabbit anti-IL-1β (1:500) (Novus Biologicals, NB600–633), rabbit anti-TNF-α (1:200) (Novus Biologicals, NBP1–19532), mouse anti-CCL2 (1:50) (Novus Biologicals, MAB28171), and goat anti-IL-6 (1:40) (R&D, AF1609) antibodies. Nuclear pSmad3-, NFκB p50-, or pH2AX-positive cells were quantified as the percent of positive cells per total epithelial cell count (excluding sloughed epithelial cells induced by irradiation). Nuclear Ki67 was quantified as positive cells per length of basement membrane. For pH2AX staining, cells with more than 3 nuclear foci were defined as pH2AX-positive cells. Myeloperoxidase-positive cells were quantified per square millimeter epithelial and stroma area above the muscle layer, and sequential 10 × images along the basement membrane were quantified and averaged per sample using Olympus cellSens Dimension software.

Paraffin-embedded sections were processed as described previously (17 (link)). Sections (5 µm) were stained with hematoxylin and eosin or with Sirius red. Pictures were taken from random fields from each section at a final magnification of ×10. Staining area was calculated by pixel counting with National Institutes of Health (NIH) ImageJ. For fluorescence microscopy, fixed livers and lungs were transferred to 30% sucrose in PBS overnight, embedded in optimum cutting temperature compound, and then cryosectioned at 5 µm. Cryosections were permeabilized and blocked with 0.3% Triton X-100 and 3% BSA in PBS. Sections were incubated with primary antibodies (rabbit anti-pSmad3, Epitomics, 1880-1, 1:100; rat anti-PDGFRβ, eBioscience, 14-1402, 1:100) overnight at 4 °C and then with fluorophore-conjugated secondary antibodies (Invitrogen). Confocal imaging was performed on a Zeiss LSM 5 Pascal microscope. pSmad immunofluorescence staining was quantified as described (32 (link)).