Purkinje cell loss in iNgly1 ^−/− mice and an NGLY1 patient. (A and B) IHC staining of calbindin in cerebella of 6-mo-old iNgly1^−/− and Ngly1^fl/fl mice. Scale bar, 500 μm (upper), 100 μm (lower). Quantification of calbindin-immunoreactive Purkinje cells is shown in B. Data are shown as the mean ± SEM. Unpaired Student’s t test. ***P < 0.001. (C and D) Representative images of H&E staining (C) and calbindin IHC-DAB staining (D) of cerebella of a NGLY1 deficiency patient and an age-matched healthy individual. Scale bar, 500 μm (left), 100 μm (right). Note: cerebellar atrophy in NGLY1 deficiency patient (low magnification in D). (E and F) Fluorescent IHC staining of cleaved caspase-3 (cl-Casp3, green) and calbindin (red) in cerebella of 6-wk-old iNgly1^−/− and Ngly1^fl/fl mice. Scale bar, 50 μm. Quantification of cl-Casp3⁺ Purkinje cells per midsagittal section is shown in F. Data are the mean ± SEM of iNgly1^−/− (n = 12) and Ngly1^fl/fl (n = 12) mice. Unpaired Student’s t test. ***P < 0.001. (G and H) Fluorescent IHC staining of mitochondrial marker COX IV (green) and calbindin (red) in cerebella of 6-wk-old iNgly1^−/− and iNgly1^fl/fl control mice. Scale bar, 25 μm. Quantification of COX IV fluorescence intensity in Purkinje cell soma is shown in H. Data are the mean ± SEM of at least 18 Purkinje cells of iNgly1^−/− (n = 6) and Ngly1^fl/fl (n = 4) mice. Unpaired Student’s t test. ***P < 0.001.