anti cxcl12 sdf 1  (R&D Systems)

Journal: Cell reports

Article Title: Post-stroke hippocampal neurogenesis is impaired by microvascular dysfunction and PI3K signaling in cerebral amyloid angiopathy

doi: 10.1016/j.celrep.2024.114848

Figure Lengend Snippet: (A) Brain hippocampal tissue WT MCAO ( n = 3 mice) and 5xFAD MCAO mice ( n = 2 mice) at 12 months of age and 7 days post MCAO were subjected to scRNA-seq. Diagram showing an overview of the scRNA-seq experiment. The contralateral hippocampus was used as an internal sham control for all analyses. (B) UMAP visualization of 18,186 isolated cells at 0.2 resolution from 12-month-old mice ( n = 2 or 3 mice per group). Similar clusters are merged to show the annotated cell types within the same cluster. AC, astrocytes; LC, leukocytes; MC, myeloid cells; Neu, neurons; NPC, neural progenitor cells; OL, oligodendrocytes; PC, pericytes; PVM, perivascular macrophages. (C) UMAP plots with each cell cluster highlighted in purple corresponding to a ubiquitous marker used to identify that cluster. (D) Heatmap revealing the scaled expression of DEGs in ECs from WT MCAO and 5xFAD MCAO mice. (E) Volcano plot depicting the top upregulated and downregulated genes in EC population in WT MCAO compared to 5xFAD MCAO. Genes that are significant at a p value ≤0.05 and log fold change ≥1.25 are portrayed in red. (F) GO pathway analysis for upregulated (yellow) or downregulated (purple) genes in the EC cluster of 5xFAD MCAO mice. Bars show −log10( p ). (G) Violin plots showing the mean and variance differences between WT MCAO and 5xFAD MCAO ECs for genes regulating blood vessel morphogenesis ( Sox18, ID1 ) regulation of Neu death ( C1QA, TYROBP, CD200 ), and chemokine signaling ( Cxcl12, Ccl4 ).

Article Snippet: Primary antibodies and dilutions were applied the next day as follows: anti-ionized calcium-binding adapter molecule 1 (Iba1; 1:1000; Fisher Scientific), neuronal nuclei (NeuN; 1:500 Abcam), anti-glial fibrillary protein (GFAP; 1:1000; 3670 Cell Signaling), Lycopersicon Esculentum (Tomato) Lectin (LEL, TL), DyLight 488 (1:200; Vector Laboratories) anti-Claudin-5 (claudin-5; 1:500 Cell Signaling), anti-Occludin (Occln; 1:500 Cell Signaling), anti-zonula occludens-1 (ZO-1; 1:500 Fisher Scientific), anti-doublecortin (DCX; 1:200 Santa Cruz), anti-BrdU (1:200, Abcam), and anti-CXCL12/SDF-1 (Cxcl12; 1:500 RD Systems).

Techniques: Control, Isolation, Marker, Expressing

Journal: Cell reports

Article Title: Post-stroke hippocampal neurogenesis is impaired by microvascular dysfunction and PI3K signaling in cerebral amyloid angiopathy

doi: 10.1016/j.celrep.2024.114848

Figure Lengend Snippet: (A) Volcano plot illustrating the downregulated genes in the NPC cluster from 5xFAD MCAO mice compared to 5xFAD sham mice. Genes significant at a p value <0.05 and log fold change ≥1.25 are portrayed in red. (B) Violin plots showing the mean and variance differences between 5xFAD MCAO and 5xFAD sham NPCs for genes regulating Neu projection guidance ( Cxcl12, Scn1b ), PI3K/AKT signaling pathway ( Pik3c2a, Creb3l2 ), and RNA processing ( Ddx23, Kin ). (C) GO pathway analyses for downregulated genes in the NPC population of the 5xFAD MCAO mice. Bars show −log10( p ). (D) Brain sections from 5xFAD sham or 5xFAD MCAO mice (sham, n = 3 mice; MCAO, n = 3 mice) at 12 months of age were subjected to co-immunostaining for Cxcl12 (red) and CD31 (green), or DCX (blue), NeuN (green), and DAPI (cyan). (E) The ratio of Cxcl12/CD31 signal was quantified. ** p = 0.0011, two-tailed Student’s t test. Scale bar, 50 μm. (F) The relative fluorescent count signal of DCX per DAPI+ cell was quantified from brain sections of mice. * p = 0.0223. (G) Incucyte ZOOM analysis of NPC neurogenesis over 16 h. ReN cells were exposed to siRNA-CXCL12, siRNA-PIK3C2A, siRNA-CREB3L2, or scrambled siRNA for 24 h before imaging in the incucyte. Representative images from time points 0 and 16 h after siRNA transfection were taken, where blue indicates neurite extensions and green represents cell bodies. Scale bar, 100 μm. (H) Quantification of Incucyte images demonstrates the change in rate of neurite length per cell-body cluster area. Graphs represent the average of individual pictures taken in 16 image grids over 16 h. Additionally, t = 0 h corresponds to the first image taken after a 24-h transfection with siRNA on the graph. xy plot is mean ± SEM. ** p = 0.0058, *** p = 0.0007, **** p < 0.0001; ††† p = 0.0001, †††† p < 0.0001; ‡‡‡‡ p < 0.0001; #### p < 0.0001; ordinary two-way ANOVA with multiple comparisons.

Article Snippet: Primary antibodies and dilutions were applied the next day as follows: anti-ionized calcium-binding adapter molecule 1 (Iba1; 1:1000; Fisher Scientific), neuronal nuclei (NeuN; 1:500 Abcam), anti-glial fibrillary protein (GFAP; 1:1000; 3670 Cell Signaling), Lycopersicon Esculentum (Tomato) Lectin (LEL, TL), DyLight 488 (1:200; Vector Laboratories) anti-Claudin-5 (claudin-5; 1:500 Cell Signaling), anti-Occludin (Occln; 1:500 Cell Signaling), anti-zonula occludens-1 (ZO-1; 1:500 Fisher Scientific), anti-doublecortin (DCX; 1:200 Santa Cruz), anti-BrdU (1:200, Abcam), and anti-CXCL12/SDF-1 (Cxcl12; 1:500 RD Systems).

Techniques: Immunostaining, Two Tailed Test, Imaging, Transfection

Journal: Cell reports

Article Title: Post-stroke hippocampal neurogenesis is impaired by microvascular dysfunction and PI3K signaling in cerebral amyloid angiopathy

doi: 10.1016/j.celrep.2024.114848

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Primary antibodies and dilutions were applied the next day as follows: anti-ionized calcium-binding adapter molecule 1 (Iba1; 1:1000; Fisher Scientific), neuronal nuclei (NeuN; 1:500 Abcam), anti-glial fibrillary protein (GFAP; 1:1000; 3670 Cell Signaling), Lycopersicon Esculentum (Tomato) Lectin (LEL, TL), DyLight 488 (1:200; Vector Laboratories) anti-Claudin-5 (claudin-5; 1:500 Cell Signaling), anti-Occludin (Occln; 1:500 Cell Signaling), anti-zonula occludens-1 (ZO-1; 1:500 Fisher Scientific), anti-doublecortin (DCX; 1:200 Santa Cruz), anti-BrdU (1:200, Abcam), and anti-CXCL12/SDF-1 (Cxcl12; 1:500 RD Systems).

Techniques: Plasmid Preparation, Recombinant, Marker, Control, Saline, Lysis, Protease Inhibitor, Stripping Membranes, Purification, Bicinchoninic Acid Protein Assay, RNAscope, Multiplex Assay, Negative Control, Software, Staining, Microscopy