Validation of Osm and Osmr expression and ligand–receptor analysis of other potential interactions between macrophages and astrocytes/fibroblasts. (A) Left column: Histological validation of Osm expression in myeloid cells at all time points after injury. Spinal cord sections were assessed by in situ hybridization for Itgam mRNA (also known as CD11b; green puncta) to label myeloid cells and Osm mRNA (white puncta). DAPI (blue) denotes cell nuclei. White arrowheads indicate CD11b⁺/Osm⁺ nuclei. Dotted white box represents the corresponding region of the magnified images on the right. Right column: Histological validation of oncostatin M receptor expression in astrocytes and fibroblasts at all time points after injury. Spinal cord sections were assessed by immunohistochemistry for GFAP (red) to label astrocytes, PDGFRβ (green) to label fibroblasts, and OSMR (white). Scale bar, 15 µm. (B) Quantification of Osm expression in myeloid (DAPI⁺/Itgam⁺/Osm⁺) and nonmyeloid (DAPI⁺/Itgam⁻/Osm⁺) cells shows significantly higher Osm expression in myeloid cells. (C and D) Quantification showed increasing colocalization of OSMR with PDGFRβ (C) and GFAP (D) immunoreactivity over time. Error bars represent SEM. *, P < 0.05 compared with uninjured tissue. One-way ANOVA and Tukey’s post hoc test. Each data point represents a biological replicate. (E) Dot plot of interaction scores for select ligand–receptor pairs at 3 and 7 dpi. Data are segregated to show interactions unique between macrophages and astrocytes (red bracket on top), unique between macrophages and fibroblasts (green bracket on bottom), or common between astrocytes and fibroblasts with macrophages (blue bracket). Macrophage interactions were split further by chemotaxis-inducing (Chemo) or Inflammatory (Inflam) macrophage subtypes. Astro, astrocyte; Fibro, fibroblast; Macro, macrophage; uninj, uninjured.