Fig. 2

Microglial cell density, pTDP-43, and GFAP load are increased in ALS. Microglial cell density was quantified from round 1 immunolabelling (A and B) and pTDP-43 load and astrogliosis was quantified from round 2 (C–F). Examples of immunofluorescent images and binary masks are taken from stage 4 ALS case, MN13 (A, C, and E). Total microglia were identified by creating separate microglial marker binary masks from HLA-DR, CD68, CD74, and Iba1, which were then combined to create a microglial marker master mask (A). Each object within this mask was considered a single microglial cell. The microglial cell density in the motor cortex and hippocampus was quantified and compared between control and ALS cases (B). Aggregates of pTDP-43 were detected, and binary masks of immunoreactivity were generated (C). The integrated intensity of pTDP-43 aggregates identified was quantified and normalised to total tissue are to generate a load measure. The pTDP-43 load was compared between control and ALS motor cortex and hippocampus (D). The area of GFAP immunoreactivity identified was quantified and normalised to total tissue are to generate a measure of astrogliosis (E and F). Data presented as mean ± SD; control n = 10 and ALS n = 9–10. Microglial densities and pathology loads 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 ≤ 0.0001, **p ≤ 0.01, *p ≤ 0.05