Neutrophils and Th17 cells colocalize in CD. (A) Confocal microscopy analysis was performed on gut tissue section of CD patients and healthy donors for CD15⁺ (green) and RORγt⁺ (red) cells. Nuclei were counterstained with TO-PRO-3 (blue). Cropped images of CD15, RORγt, and related isotype Abs (A bottom panels), as well as of CD3, RORγt, and related isotype Abs (B) are also reported (original magnification ×400, pixel 1024 × 1024). Representative images from 3 patients and 3 healthy donors. Images were acquired with a 40×/1.3 NA oil objective (corresponding to a 400× magnification). Single confocal images of cells were obtained at nuclear equatorial level by using the 1 Airy unit formula for the adjustment of the pinhole diameter, corresponding to an optical slice of 0.7 mm (for FITC emission images). Images were acquired and analyzed using the LSM 5 software (Carl Zeiss). The slides were mounted with Vectashield mounting medium (Vector Laboratories).