Journal: Advanced Science

Article Title: A Biomimetic Norepinephrine‐Loaded Aligned Mineralized Collagen Scaffold for Coordinated Neurovascular, Osteogenic, and Immunomodulatory Repair of Critical‐Sized Bone Defects

doi: 10.1002/advs.202518807

Figure Lengend Snippet: Scaffold‐mediated neurogenic support and axonal guidance. Schematic illustration of the indirect a) and direct b) co‐culture strategies used to evaluate scaffold effects on DRG neurons. c,d) Immunofluorescence analysis of DRG neurons cultured with conditioned media. (c) TUBB3 staining at day 3. (d) TUBB3 and CGRP staining at day 6. e) TUBB3 staining of DRG neurons directly cultured on scaffold surfaces for 4 days. f) Quantification of neurite length from the indirect co‐culture model at day 3. g) qPCR analysis of Ngf expression in rBMSCs cultured with scaffolds for 3, 7, and 14 days. h) Neurite orientation analysis derived from angular measurements in panel E. Scale bars: 100 µm in (c and d), 50 µm in (e). * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. a and b Created with biorender.com. Data are mean ± SD ( n = 3).

Article Snippet: Neurite outgrowth was evaluated on day 3 via TUBB3 (Proteintech, 66375‐1‐Ig, 1:500) immunofluorescence staining, while CGRP (Bioss, bs‐10639R, 1:500) expression was assessed on day 6.

Techniques: Co-Culture Assay, Immunofluorescence, Cell Culture, Staining, Expressing, Derivative Assay

Journal: Advanced Science

Article Title: A Biomimetic Norepinephrine‐Loaded Aligned Mineralized Collagen Scaffold for Coordinated Neurovascular, Osteogenic, and Immunomodulatory Repair of Critical‐Sized Bone Defects

doi: 10.1002/advs.202518807

Figure Lengend Snippet: In vivo immunofluorescence analysis of neurovascular and immune responses at 4 and 8 weeks post‐implantation. Representative immunofluorescence images showing the expression of TUBB3 (neuronal marker), CGRP (sensory neuropeptide), VEGFA (angiogenic factor), CD31 (endothelial cell marker), CD86 (M1 macrophage marker), and IL‐10 (M2 macrophage marker) in bone defect regions at 4 (a) and 8 (b) weeks across different groups. Quantitative analysis of fluorescence intensity is shown on the right. Scale bars: 50 µm. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. Data are mean ± SD ( n = 3).

Article Snippet: Neurite outgrowth was evaluated on day 3 via TUBB3 (Proteintech, 66375‐1‐Ig, 1:500) immunofluorescence staining, while CGRP (Bioss, bs‐10639R, 1:500) expression was assessed on day 6.

Techniques: In Vivo, Immunofluorescence, Expressing, Marker, Fluorescence

Journal: Cell Death Discovery

Article Title: PGC-1a mediated mitochondrial biogenesis promotes recovery and survival of neuronal cells from cellular degeneration

doi: 10.1038/s41420-024-01953-0

Figure Lengend Snippet: A Representative bright field (BF) and immunofluorescence images of 5% EtOH treatment (vol/vol, 1 and 3 h) induced neurite retraction, and its regeneration after washing EtOH (Washed, 4 and 20 h) in neuronal PC12 cells. Green: neuron-specific β-III tubulin; Blue: DAPI. Scale bar: 50 µm. B , C Quantification results of mean neurite length per cell and the percentage of cells with neurites. *** P < 0.001, vs. control group; ## P < 0.01, ### P < 0.001, vs. EtOH 3 h group. EtOH ethanol.

Article Snippet: For immunostaining, cells were incubated with primary antibodies against neuron-specific β-III tubulin (MAB1195, R&D systems, USA), LC3B (NB100-2220, Novus Biologicals, USA), TOM20 (42406S, Cell Signaling Technology), and Drp1 (8570S, Cell Signaling Technology) overnight at 4 °C.

Techniques: Immunofluorescence, Control

Journal: Cells

Article Title: Neuronal Differentiation of GBM-Initiating Cells Combined with Elimination of Undifferentiated Cells Preserves Motor Function

doi: 10.3390/cells15060539

Figure Lengend Snippet: ISX9 induced GICs to differentiate into neurons. ( A ) Proliferation of GICs, E6 (closed circle) and E16 (open circle), cultured in the presence of various concentrations of ISX9, was determined using the MTT assay. ( B , C ) Dose-dependent ISX9 effects on βIII tubulin ( B ) and Ki67 ( C ) in GICs, E6 (closed circle), and E16 (open circle). ( D ) Heat map with hierarchical clustering of the top 1000 genes in GICs, E6, and E16, cultured with DMSO or ISX9 (30 μM) for 3 days. ( E ) Enriched pathways for clusters A and D. ( F ) Immunoreactivity of hGICs cultured in the presence of ISX9 (30 μM) for 3 days, for hSynapsin (green) and MAP2 (red). The higher magnification images are shown on the bottom right of each figure surrounded by white dotted lines. All nuclei are labeled with Hoechst33342 (Hoechst, blue). Scale bar: 20 μm. ( G ) Quantitative data of ( F ). ** p < 0.01, *** p < 0.001. Statistical significance was determined using the t -test. Error bars indicate ±SD.

Article Snippet: Primary Abs used were as follows: mouse anti-βIII tubulin (1:100, R&D Systems, Minneapolis, MN, USA, MAB1195), mouse monoclonal anti-Nestin (1:200, BD Biosciences, San Jose, CA, USA, 611658), mouse anti-human synapsin I/II/III (1:1000, hSynapsin, Biolegend, San Diego, CA, USA, 16245), mouse monoclonal anti-Epithelial-V-like antigen 1 (1:200, EVA1, clone B2E5), chicken anti-βIII tubulin (1:5000, Novus Biologicals, Centennial, CO, USA, NB100-1612), chicken anti-microtubule associated protein 2 (1:5000, MAP2, Novus Biologicals, NB300-213), rabbit anti-GFAP (1:500, Dako, Glostrup, Denmark, N1506), and rabbit anti-Ki67 (1:250, ThermoFisher, MA5-14520).

Techniques: Cell Culture, MTT Assay, Labeling

Journal: Cells

Article Title: Neuronal Differentiation of GBM-Initiating Cells Combined with Elimination of Undifferentiated Cells Preserves Motor Function

doi: 10.3390/cells15060539

Figure Lengend Snippet: Examination of optimal method that induces neurons from as many GICs as possible and eliminates undifferentiated GICs. GICs were cultured under three culture conditions, both ISX9 and BRQ simultaneously, ISX9 followed by BRQ, and BRQ followed by ISX9, with either the maximum concentration that induced neuronal differentiation or half the concentration, as shown in and previously . The surviving cells were counted ( A ) and immunolabeled for the GIC marker EVA1 ( B ), two neuronal markers, βIII tubulin ( C ) and MAP2 ( D ), and the proliferation marker Ki67 ( E ). The table shows the concentrations of the chemicals used in each experiment. The arrows indicate the sequential chemical treatments. Error bars indicate ±SD. Statistical significance was determined using one-way ANOVA. Error bars indicate ±SD.

Article Snippet: Primary Abs used were as follows: mouse anti-βIII tubulin (1:100, R&D Systems, Minneapolis, MN, USA, MAB1195), mouse monoclonal anti-Nestin (1:200, BD Biosciences, San Jose, CA, USA, 611658), mouse anti-human synapsin I/II/III (1:1000, hSynapsin, Biolegend, San Diego, CA, USA, 16245), mouse monoclonal anti-Epithelial-V-like antigen 1 (1:200, EVA1, clone B2E5), chicken anti-βIII tubulin (1:5000, Novus Biologicals, Centennial, CO, USA, NB100-1612), chicken anti-microtubule associated protein 2 (1:5000, MAP2, Novus Biologicals, NB300-213), rabbit anti-GFAP (1:500, Dako, Glostrup, Denmark, N1506), and rabbit anti-Ki67 (1:250, ThermoFisher, MA5-14520).

Techniques: Cell Culture, Concentration Assay, Immunolabeling, Marker

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