Fig. 2
Brains infected with Cn strain H99 cells exhibit initial increased activated microglia cell density at 3-dpi. (A) Immunofluorescent images of brain tissue sections harvested 3-dpi from naïve, saline-injected, H99-infected, and cap59-infected CX3CR1-EGFP mice (n = 4 mice per group; 104 fungi). EGFP (green) is expressed by microglia. Calcofluor white was used to label the cell wall of acapsular or capsular yeast cells (blue). GXM-specific mAb 18B7 was used to stain H99 cell capsular- or exo-polysaccharide (red). Left, central, and right panels show 10, 20, and 40X magnification, respectively. Scale bars: 100 μm (10X and 20X) and 50 μm (40X). The 20X images in each group were randomly analyzed (n = 10 fields per group) for microglia cell (B) density per µm2, (C) nearest neighbor index (NNI), (D) clusters per µm2, and (E) spacing index (NNI2 x microglia cell density) using the ImageJ FIJI software and NNI plugin. For B-E, bars and error bars denote mean values and SDs, respectively. Significance (****, P < 0.0001; **, P < 0.01; *, P < 0.05) was calculated by one-way ANOVA and adjusted using Tukey’s post-hoc analysis. ns denotes comparisons which are not statistically significant. This experiment was performed twice, similar results were obtained each time, and all the results combined are presented