Fig. 4.

Validation of RRIMs in ALS mouse models using in situ hybridization. (A) Fresh-frozen spinal cords from 3.5-mo-old WT and SOD1^G93A littermates treated with either vehicle or Nec-1s for 4 wk were sectioned and prepared using RNAscope with the indicated probes, followed by confocal imaging. Representative images from n = 3 mixed gender animals per group; n = 5 to 6 images from ventral horn (1 image per section). (Scale bar, 5 μm.) (B) Fresh-frozen spinal cords from 3.5-mo-old Optn^Fl/Fl, Optn^−/−, and Optn^−/−;Ripk1^D138N animals were sectioned and prepared using RNAscope with the indicated probes, followed by confocal imaging. Representative images from n = 3 mixed gender animals per group; n = 5 to 6 images from ventral horn (1 image per section). (Scale bar, 5 μm.) (C) Quantification of (A) average of 14 microglia analyzed per image using CellProfiler. n = 3 mixed gender animals per group; n = 5 to 6 images from ventral horn (1 image per section). Two-way ANOVA with multiple comparison testing relative to SOD1^G93A Vehicle group. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. Data are represented as mean ± SEM. (D) Quantification of (B) average of 17 microglia analyzed per image using CellProfiler. n = 3 mixed gender animals per group; n = 5 to 6 images from ventral horn (1 image per section). Two-way ANOVA with multiple comparison testing relative to Optn^−/− Vehicle group. ****P < 0.0001; ns, not significant. Data are represented as mean ± SEM.