Characterization of Ngly1-deficient mice. (A) H&E and Luxol fast blue analysis of the thoracic spinal cord of 6-mo-old iNgly1^−/− and iNgly1^fl/fl control mice. The data are representative of at least three independent mice per genotype. (B and C) Innervation (B) and synaptic size (C) of E18.5 Ngly1^−/− and littermate fetus (n = 2 per genotype). α-Bungarotoxin was used to label postsynaptic ACh receptors in the muscle (red), and antibody against syntaxin was used to label presynaptic nerves (green). Low-power (10×) views of the phrenic nerve/diaphragms (B); scale bar, 2 mm. Note: the images were rotated after acquisition for presentation to maintain consistent anatomical orientation across samples. Nontissue triangular regions in the corners were outlined with dashed white lines. High-power (63×) views of neuromuscular junctions in the diaphragm (C); scale bar, 20 μm. Presynaptic nerve terminal, green; postsynaptic AChRs, red. (D) Representative fluorescent IHC staining of Purkinje cell marker calbindin (green) in cerebella of 5-wk-old JF1/BL6 isogenic F1 Ngly1^−/− and Ngly1^+/+ control mice. n = 3 per genotype. Scale bar, 500 μm (left), 100 μm (right). (E) Quantification of calbindin-immunoreactive Purkinje cells shown in D. Data are shown as the mean ± SEM. Student’s t test. *P < 0.05. (F) Representative IHC staining of NGLY1 in cerebellum (left) and cerebral cortex (right) of cynomolgus macaque monkeys. Scale bar, 500 μm (zoom-out), 100 μm (zoom-in). (G) Multiplex cytokine analysis of female and male iNgly1^−/− (n = 10 females, 9 males) and Ngly1^fl/fl (n = 8 females, 8 males) mouse serum.