Fig. 2.

Alternatively activated neutrophils localize to inflammatory spinal cord lesions and are positioned adjacent to T cells. Confocal maximum-intensity projections (MIPs) of spinal cord sections obtained from 2 representative mice at clinical EAE onset. Sections were immunofluorescently labeled for Arg1 (green), Ly6G (magenta), CD3ε (orange), and DAPI (blue), and probed for Cxcr2 mRNA expression by RNAscope (white). Orange boxes in the schematics indicate the anatomical locations of the imaged regions; white boxes denote regions from which single optical sections are shown below. Single optical sections from the boxed regions are shown as merged images and corresponding single channels (DAPI, ARG1, CD3ε, Ly6G, and Cxcr2). Dashed outlines indicate alternatively activated neutrophils (aaN; CD3ε⁻Ly6G⁺/⁻Cxcr2⁺Arg1⁺; green), classically activated neutrophils (N; CD3ε⁻Ly6G⁺Cxcr2⁺Arg1⁻; magenta), and T cells (CD3ε⁺Ly6G⁻Cxcr2⁻; orange). Images were acquired at 40 × original magnification. A Single optical section from the MIP on the left side showing two Arg1⁺Ly6G⁺ aaN in close proximity to a CD3ε⁺ T cell; an Arg1⁻ conventional neutrophil (N) is also present. B Single optical section from the MIP on the right side highlighting an Arg1^high Ly6G^low Cxcr2⁺ aaN. Images are representative of 6 mice from 2 independent experiments; 9 sections were analyzed in total