SMAD3-KO granulocytes cause tissue damage in the colon. (A) Representative CD11b staining and (B) average CD11b geometric mean fluorescence intensity of granulocytes extracted from the colon of AHCT recipients on day 40 (mean and SEM, n = 9). (C) Representative images (following Visiopharm software analysis) of consecutive tissue sections stained for Gr-1 (red) or nitrotyrosine (blue); overlay created using the Adobe Photoshop, Version CS3 software. Representative images were taken from virtual slides that were scanned with the NanoZoomer, Version 2.0 series system and acquisition software NDP.scan, Version 2.2.17 (Hamamatsu Photonics) using a 40 × objective lens. Analysis of positively stained cells (green) was done using Visiopharm Integrator System, Version 3.4.1.0 MicroImager (Visiopharm) software. Overlay analysis of consecutive tissue sections stained for Gr-1 or nitrotyrosine were photomerged and analyzed using the Adobe Photoshop, Version CS3 software. (D) Percentage of Gr-1⁺ and nitrotyrosine⁺ cells (mean and SEM, n = 3; 10 fields at a 20× original magnification). (E) Percentage of nitrotyrosine-positive cells that are adjacent to granulocytes (≤ 30 pixels). (F) Average weight (with SEM) for surviving mice (minimum of 6 mice per group) at day 40 after AHCT. (G) Pathologic grading of large bowel GVHD (n = 6-9). Statistical analyses were performed with Student t test: *P < .05, **P < .01.