Lack of neuroinflammation in iNgly1 ^−/− mice. (A–C) Heatmap of mRNA expression in cerebella of iNgly1^−/− (n = 7) and iNgly1^fl/fl control (n = 9) mice. mRNA was measured by RT-qPCR array. Bar graphs are shown as the mean ± SEM. Unpaired Student’s t test. **P < 0.01; ***P < 0.001; ns, not significant. (D) Representative fluorescent IHC staining of microglial marker IBA1 (red) in cerebella of 3-mo-old iNgly1^−/− and iNgly1^fl/fl control mice. The nucleus was stained with DAPI (blue). Scale bar, 2 mm (left), 500 μm (right). Images were acquired using Zeiss AxioScan with automated stitching. (E) Quantification of microglia numbers (IBA1, red in D) in the FOV of the cerebellar molecular layer of iNgly1^−/− (n = 13) and Ngly1^fl/fl (n = 11) mice. Data are the mean ± SEM. Unpaired Student’s t test, ns, not significant. (F) Representative fluorescent IHC staining of microglial marker IBA1 (green) in cerebella of 5-wk-old C57BL/6-JF1 isogenic F1 Ngly1^−/− and Ngly1^+/+ control mice. Scale bar, 500 μm. Images were acquired using Hamamatsu NanoZoomer with automated stitching. (G) Quantification of microglial numbers (IBA1, green in F) in the FOV of the cerebellar molecular layer of C57BL/6-JF1 isogenic F1 Ngly1^−/− (n = 3) and Ngly1^+/+ (n = 3) mice. Data are the mean ± SEM. Unpaired Student’s t test, ns, not significant. (H) Representative IHC staining of microglial marker IBA1, activation marker CD68, and astrocyte marker GFAP in cerebella of 6-mo-old iNgly1^−/− and iNgly1^fl/fl control mice. The data were verified in at least three mice. Scale bar, 50 μm (IBA1 upper, CD68 upper, and GFAP), 10 μm (IBA1 lower, CD68 lower). FOV, field of view.