Journal: JCI Insight

Article Title: Gut mucosal cells transfer α -synuclein to the vagus nerve

doi: 10.1172/jci.insight.172192

Figure Lengend Snippet: ( A ) Intestinal organoids were prepared from an SNCA A53T mouse in which CCK-containing cells express enhanced green fluorescent protein (eGFP), and vagal nodose ganglia neurons were isolated from an Snca –/– mouse lacking endogenous α-synuclein. ( B ) Representative images of organoids and neurons grown in coculture for 5 days, with eGFP-positive cells (green) in the organoid and β-tubulin III (Tuj1, cyan) highlighting neuronal processes. ( C ) Representative high-magnification α-synuclein (red) staining of an eGFP-positive EEC. Red arrow indicates localization to a PGP9.5-positive (cyan) process in an Snca –/– mouse neuron. ( D and E ) Representative images with neuron-specific β-tubulin III (cyan). Surface and (adjacent) intracellular confocal slices are shown. Scale bars are 30 μm for B , 3 μm for C , and 5 μm for D and E .

Article Snippet: Primary antibodies used for immunostaining included rabbit CCK , rabbit α-synuclein (Abcam catalog ab138501, RRID:AB_2537217, at 1:1,000), guinea pig PGP9.5 (Abcam catalog ab10410, RRID:AB_297150, at 1:100), chick β-tubulin III (Tuj1; Neuromics catalog CH23005, RRID:AB_2210684, at 1:100), and chick GFP (Abcam catalog ab13970, RRID:AB_300798, at 1:1,000).

Techniques: Isolation, Staining

Journal: Cell Death & Disease

Article Title: ALS-related human cortical and motor neurons survival is differentially affected by Sema3A

doi: 10.1038/s41419-018-0294-6

Figure Lengend Snippet: a RT-PCR analysis for 1-PlexinA1, 2-PlxinA2, 3-PlxinA3, 4-PlxinA4, 5-NRP1, 6-NRP2, 7-GAPDH, and M-DNA ladder 100 bp. b , c MNs ( b ) and cortical neurons ( c ) were immunostained and imaged at days 30. Representative confocal microscopy images are shown (scale bars = 20 µm ( b ) and bars = 25 µm ( c ).

Article Snippet: The following proteins were evaluated: Tbr1 (ab31940, Abcam, 1:500), HB9 (81.5C10, DSHB, 1:80), CHAT (AB144P, Millipore-Merck, 1:200), Olig2, β3-Tubulin (Sigma T8578, 1:1000), (AF2418, R&D, 1:80), Anti-NRP1 (GTI5036, Neuromics, 1:200).

Techniques: Reverse Transcription Polymerase Chain Reaction, Confocal Microscopy

Journal: Cell Death & Disease

Article Title: ALS-related human cortical and motor neurons survival is differentially affected by Sema3A

doi: 10.1038/s41419-018-0294-6

Figure Lengend Snippet: a Cell-electrode impedance assay indicates response of U87MG cells to Sema3A. b Percentage of HB9-GFP MNs was analyzed by FACS 72 h after treatment with Sema3A, Sema3A with NRP1 blocking antibody or control media. MNs were gated as PI-negative and GFP-positive relative to GFP-negative control cells. Data are normalized to control treatment and represented as mean of three independent experiment ± SEM. P value calculated by unpaired t test. c Quantification of imaged HB9-GFP-positive MNs 48 h after treatment with Sema3A or control media. Data are represented as mean ± SEM of four independent experiments. P value calculated by unpaired t test. d Quantification of immunostained and imaged MNs positive for GFP and cleaved caspase-3. Data are represented as mean ± SEM of three independent experiments. e Quantification of immunostained and imaged MNs positive for CHAT and cleaved caspase-3. Data are represented as mean ± SEM of three independent experiments. P value calculated by unpaired t test.

Article Snippet: The following proteins were evaluated: Tbr1 (ab31940, Abcam, 1:500), HB9 (81.5C10, DSHB, 1:80), CHAT (AB144P, Millipore-Merck, 1:200), Olig2, β3-Tubulin (Sigma T8578, 1:1000), (AF2418, R&D, 1:80), Anti-NRP1 (GTI5036, Neuromics, 1:200).

Techniques: Blocking Assay, Negative Control

Journal: Cell Death & Disease

Article Title: ALS-related human cortical and motor neurons survival is differentially affected by Sema3A

doi: 10.1038/s41419-018-0294-6

Figure Lengend Snippet: a The percentage of cortical neurons was analyzed by FACS 72 h after treatment with Sema3A, Sema3A with NRP1 blocking antibody or control media. Cortical neurons were immunostained and gated as PI-negative and Tbr1-positive relative to unstained control cells. Data are normalized to control treatment and represented as mean of three independent experiment ± SEM. P value calculated by unpaired t test. b Quantification of imaged cortical neurons stained for Tbr1 after 72 h treatment with Sema3A and control media. c Quantification of immunostained and imaged cortical neurons positive for Tbr1 and cleaved caspase-3. Data are represented as mean ± SEM of three independent experiments.

Article Snippet: The following proteins were evaluated: Tbr1 (ab31940, Abcam, 1:500), HB9 (81.5C10, DSHB, 1:80), CHAT (AB144P, Millipore-Merck, 1:200), Olig2, β3-Tubulin (Sigma T8578, 1:1000), (AF2418, R&D, 1:80), Anti-NRP1 (GTI5036, Neuromics, 1:200).

Techniques: Blocking Assay, Staining

Journal: Developmental biology

Article Title: Galnt17 loss-of-function leads to developmental delay and abnormal coordination, activity, and social interactions with cerebellar vermis pathology

doi: 10.1016/j.ydbio.2022.08.002

Figure Lengend Snippet: (A) Tuj1 (left) and CalB (right) staining showed the morphology of Purkinje cells in WT (top) and Galnt17−/− (bottom) animals. Galnt17−/− mutants lost thick primary branches (arrowheads), and the primary branches were bifurcated (arrows) compared to WT mice at P60. (B) Galnt17−/− mutants (bottom) showed fewer CalB (green) marked axons in axonal tract in lobule IV/V compared to WT mice (top) at P14. (C) Width of axonal tract was measured, and it showed mutants (dark gray) have narrower axonal tract than WT (light gray) in lobule IV/V at P14. n=3 for each genotype. t-Test, *p<0.05. (D) Galnt17−/− mutants (right) showed forelimb clasping (arrowhead) during tail suspension test compared to WT (left) mice in adult. n=5 animals for each genotype were scored for clasping (1) or no clasping (0) with all mutant animals scoring as 1 and all WT animals scoring as 0 in this test. Scale bar = 50 μm.

Article Snippet: Primary antibodies: GFAP (Invitrogen 180063, 1:400), BrdU (Abcam ab6326, 1:400), Calbindin (CalB; Sigma C9848, 1:400), beta-III Tubulin (Tuj1; Neuromics CH23005, 1:200), Phospho-H3 (Millipore 06–570, 1:500); secondary antibodies: Thermo-Fisher Scientific, Alexa Fluor 488, Alexa Fluor 594.

Techniques: Staining, Suspension, Mutagenesis