Fig. 3

Microglia increase expression of CD68 and Iba1 in the ALS motor cortex. Microglia were identified by combined labelling of all microglial markers from round 1 and the tissue-wide and single cell average intensities of each of these markers was measured and used to quantify changes in functional marker expression in ALS; L-ferritin (A–C), HLA-DR (D–F), CD68 (G–I), CD74 (J–L), and Iba1 (M–O). Immunolabelling intensity of each functional marker was measured within the microglial master mask and normalised to the area of the microglial master mask. The tissue-wide average intensities were compared between control and ALS cases in the motor cortex and hippocampus for each functional marker (A, D, G, J, and M). Data presented as mean ± SD; control n = 10 and ALS n = 9–10. To designate cells as either high- or low-expressing for each functional marker of interest (MOI^high or MOI^low), total microglia from all control and all ALS cases were pooled and the distribution curve for each marker was generated (B, E, H, K, and N). The threshold for each marker was determined and each cell was designated as either MOI^high or MOI^low for each functional marker. The percentage of MOI^high cells was compared between control and ALS cases in the motor cortex and hippocampus for each functional marker (C, F, I, L, and O). Data presented as mean ± SD; control n = 10 and ALS n = 9–10. MOI average intensities and MOI^high percentages were compared between case groups with multiple Mann–Whitney tests and multiple comparisons were controlled for using a False Discovery Rate of 0.01, as determined by the two-stage step-up method of Benjamini, Krieger, and Yekutieli. Significance of differences between case groups: *p ≤ 0.05, ** p ≤ 0.01, ***p ≤ 0.001