rabbit anti calbindin d28k  (Proteintech)

Journal: The Journal of Experimental Medicine

Article Title: The STING pathway drives noninflammatory neurodegeneration in NGLY1 deficiency

doi: 10.1084/jem.20242296

Figure Lengend Snippet: Characterization of Ngly1 -deficient mice. (A) H&E and Luxol fast blue analysis of the thoracic spinal cord of 6-mo-old i Ngly1 −/− and i Ngly1 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 i Ngly1 −/− ( n = 10 females, 9 males) and Ngly1 fl/fl ( n = 8 females, 8 males) mouse serum.

Article Snippet: DAB immunohistochemistry of calbindin (Calbindin [D1I4Q] XP Rabbit mAb, cat #13176; Cell Signaling) was performed at HistoWiz.

Techniques: Control, Immunohistochemistry, Marker, Multiplex Assay

Journal: The Journal of Experimental Medicine

Article Title: The STING pathway drives noninflammatory neurodegeneration in NGLY1 deficiency

doi: 10.1084/jem.20242296

Figure Lengend Snippet: Purkinje cell loss in i Ngly1 −/− mice and an NGLY1 patient. (A and B) IHC staining of calbindin in cerebella of 6-mo-old i Ngly1 −/− 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 i Ngly1 −/− 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 i Ngly1 −/− ( 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 i Ngly1 −/− and i Ngly1 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 i Ngly1 −/− ( n = 6) and Ngly1 fl/fl ( n = 4) mice. Unpaired Student’s t test. ***P < 0.001.

Article Snippet: DAB immunohistochemistry of calbindin (Calbindin [D1I4Q] XP Rabbit mAb, cat #13176; Cell Signaling) was performed at HistoWiz.

Techniques: Immunohistochemistry, Staining, Marker, Control, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: The STING pathway drives noninflammatory neurodegeneration in NGLY1 deficiency

doi: 10.1084/jem.20242296

Figure Lengend Snippet: Deletion of Ngly1 in Purkinje cells or microglia alone did not cause neurodegeneration. (A) Representative IHC staining of Purkinje cell marker calbindin in cerebella of 6-mo-old Ngly1 fl/fl , Ngly1 fl/fl Pcp2-cre , and Ngly1 fl/fl Cx3cr1-cre control mice. Scale bar, 1 mm (upper panel), 100 μm (lower panel). (B) Quantification of calbindin-immunoreactive Purkinje cells shown in A. Data are shown as the mean ± SEM. Unpaired Student’s t test. ns, not significant.

Article Snippet: DAB immunohistochemistry of calbindin (Calbindin [D1I4Q] XP Rabbit mAb, cat #13176; Cell Signaling) was performed at HistoWiz.

Techniques: Immunohistochemistry, Marker, Control

Journal: The Journal of Experimental Medicine

Article Title: The STING pathway drives noninflammatory neurodegeneration in NGLY1 deficiency

doi: 10.1084/jem.20242296

Figure Lengend Snippet: Sting1 −/− ameliorates neurological disease of i Ngly1 −/− mice. (A) Schematic diagram showing generation of i Ngly1 −/− Sting1 −/− mice. (B) Survival curves of Ngly1 fl/fl , Ngly1 fl/fl Sting1 −/− , i Ngly1 −/− , and i Ngly1 −/− Sting1 −/− male mice. Log-rank (Mantel–Cox) test. **P < 0.01; ***P < 0.001. (C) Neurological deficit score of Ngly1 fl/fl , Ngly1 fl/fl Sting1 −/− , i Ngly1 −/− , and i Ngly1 −/− Sting1 −/− male mice. Data were shown as median ± 95% CI. Mann–Whitney test. (D) IHC staining of calbindin in cerebella of 1-year-old Ngly1 fl/fl , Ngly1 fl/fl Sting1 −/− , i Ngly1 −/− , and i Ngly1 −/− Sting1 −/− mice. Scale bar, 500 μm (upper), 100 μm (lower). (E) Quantification of Purkinje cell number of indicated genotypes. Data were shown as the mean ± SEM. One-way ANOVA with Bonferroni’s multiple comparisons test. **P < 0.01; ***P < 0.001. (F and G) IHC staining of cleaved caspase-3 (cl-Casp3, green) and calbindin (red) in cerebella of 8-wk-old Ngly1 fl/fl , Ngly1 fl/fl Sting1 −/− , i Ngly1 −/− , and i Ngly1 −/− Sting1 −/− mice. Scale bar, 50 μm. Quantification of cl-Casp3 + Purkinje cells per midsagittal cerebellar section is shown in G. Data are from mice ( n = 12 per genotype) of three independent experiments. Data were shown as the mean ± SEM. One-way ANOVA with Bonferroni’s multiple comparisons test. ***P < 0.001.

Article Snippet: DAB immunohistochemistry of calbindin (Calbindin [D1I4Q] XP Rabbit mAb, cat #13176; Cell Signaling) was performed at HistoWiz.

Techniques: MANN-WHITNEY, Immunohistochemistry

Journal: The Journal of Experimental Medicine

Article Title: The STING pathway drives noninflammatory neurodegeneration in NGLY1 deficiency

doi: 10.1084/jem.20242296

Figure Lengend Snippet: Pharmacological inhibition of STING ameliorates neurological disease of NGLY1 deficiency. (A) Experimental workflow of treatment of i Ngly1 −/− mice. (B) Neurological deficit score of Ngly1 fl/fl ( n = 7 mice) and i Ngly1 −/− mice treated with STING inhibitor VS-X4 ( n = 8 mice) or vehicle control ( n = 9 mice). Data were shown as median ± 95% CI. Mann–Whitney U test. **P < 0.01; ***P < 0.001; ns, not significant. (C) Disease onset (disease incidence in the month when kyphosis score reaches 2) of Ngly1 fl/fl ( n = 7 mice) and i Ngly1 −/− mice treated with STING inhibitor VS-X4 ( n = 8 mice) or vehicle control ( n = 9 mice). Log-rank (Mantel–Cox) test. *P < 0.05. (D) IHC staining of calbindin in cerebella of i Ngly1 −/− mice treated with the STING inhibitor VS-X4 or vehicle control for 3 mo. Scale bar, 500 μm (upper), 100 μm (lower). (E) Quantification of Purkinje cell number of i Ngly1 −/− mice treated with the STING inhibitor VS-X4 or vehicle control for 3 mo. One-way ANOVA with Bonferroni’s multiple comparisons test. *P < 0.05; **P < 0.01.

Article Snippet: DAB immunohistochemistry of calbindin (Calbindin [D1I4Q] XP Rabbit mAb, cat #13176; Cell Signaling) was performed at HistoWiz.

Techniques: Inhibition, Control, MANN-WHITNEY, Immunohistochemistry