rabbit anti human anti p2y 1  (Alomone Labs)

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: The P2Y 13 receptor is expressed by qNSCs in the adult SEZ. A. The expression of the different metabotropic P2Y receptors in the adult SEZ was analyzed by quantitative RT-PCR (n=6). B. P2Y 13 receptor expression in the ventral and dorsal wall of the adult SEZ analyzed by quantitative RT-PCR (n=6). C . Comparison of the P2Y 13 receptor protein in the ventral and dorsal wall of the SEZ (n=3). D-F . P2Y 13 receptor expression in the ventral wall of the SEZ. Note how the P2Y 13 receptor (red) co-localizes with GFAP positive (white) astroglia (yellow arrowheads) but not with Ascl1 positive TAPs or cells expressing βIII-tubulin (green, scale bar 30 µm except for Ascl1 10 µm). G . Co-localization of the P2Y 13 receptor (red) in NSCs with SOX2 (white) and GFAP (green) in the adult SEZ (scale bar 30 µm). H. SEZ-derived cell populations expressing the P2Y 13 receptor in a FACs analysis using a P2Y 13 -GFP conjugated antibody. Note how the expression is mainly associated with the qNSC fraction (n=3). All graphs show the mean ±SEM: *p<0.05, **p<0.01, and ***p<0.001 (T-test).

Article Snippet: Both the cells and tissue sections were then incubated overnight at 4 °C with the primary antibodies: rabbit anti-P2Y 13 (1:100, Alomone Labs Cat# APR-009), rabbit anti-P2Y 1 (1:100, Alomone Labs Cat# APR-017), rabbit anti-SOX2 (1:100, ABclonal Cat# A0561), mouse anti-GFAP (1:200, Sigma-Aldrich Cat# G3893), mouse anti-βIII-Tubulin (1:800, Sigma-Aldrich Cat# T8660), guinea pig anti-(Doublecortin)DCX (1:400, Millipore Cat# AB2253), mouse anti-ASCL1 (1:100, BD Biosciences Cat# 556604), rabbit anti-KI67 (1:100, Fisher Scientific Cat# RM-9106-S), and chicken anti-GFP (1:400, AvesLab Cat# GFP-1020).

Techniques: Expressing, Quantitative RT-PCR, Comparison, Derivative Assay

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: Expression of the P2Y 13 receptor in the neurogenic lineage of the SEZ-derived cell cultures after 6 DIV: GFAP (magenta), Ascl1 (green, middle panel), βIII-tubulin (green, lower panel), P2Y 13 (red). Note how P2Y 13 receptor expression only co-localizes with GFAP in cells. Scale bar 30 µm.

Article Snippet: Both the cells and tissue sections were then incubated overnight at 4 °C with the primary antibodies: rabbit anti-P2Y 13 (1:100, Alomone Labs Cat# APR-009), rabbit anti-P2Y 1 (1:100, Alomone Labs Cat# APR-017), rabbit anti-SOX2 (1:100, ABclonal Cat# A0561), mouse anti-GFAP (1:200, Sigma-Aldrich Cat# G3893), mouse anti-βIII-Tubulin (1:800, Sigma-Aldrich Cat# T8660), guinea pig anti-(Doublecortin)DCX (1:400, Millipore Cat# AB2253), mouse anti-ASCL1 (1:100, BD Biosciences Cat# 556604), rabbit anti-KI67 (1:100, Fisher Scientific Cat# RM-9106-S), and chicken anti-GFP (1:400, AvesLab Cat# GFP-1020).

Techniques: Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: The P2Y 13 receptor is active in SEZ-derived NSCs in culture. NSCs in culture and loaded with the calcium dye Fura-2 were stimulated with 2MeSADP and after a washout period, they were re-exposed to 2MeSADP in the presence of either the P2Y1 receptor antagonist MRS2179 A or the P2Y 13 receptor antagonist MRS2179 B . All compounds were tested at a concentration of 10 μM and representative traces of the F340/F380 fluorescence ratios recorded from single cells are shown. C . Immunocytochemistry identifying GFAP (green) and SOX2 (red) double positive NSCs that respond to the selective agonist of the P2Y 13 receptor, 2MeSADP (yellow arrowheads). Conversely, the intracellular calcium levels of GFAP positive parenchymal astrocytes (white arrow) does not change. D . Effects of 2MeSADP on voltage-gated currents in SEZ-derived NSCs in culture. Outward currents evoked by a depolarising pulse (+10 mV, 100 ms from a Vh of −80 mV) were increased in the presence of 2MeSADP (10 µM, 2 min). Subsequent exposure to MRS221 (10 µM, 2 min) partially reversed the potentiating effect of 2MeSADP (upper panel). At variance, exposure to MRS2179 (10 µM, 2 min) of 2MeSADP with MRS2179 (10 µM, 2 min) did not have any effect on outward current increase elicited by 2MeSADP (10 µM, 2 min) (lower panel). E . Scatter plot of outward current charges from the experiments shown in ( D ), in the presence or absence of 2MeSADP (10 µM, 2 min: left panel), and following co-incubation (right panels) with MRS2211 (upper right) or MRS2179 (lower right). The values are the means ± SEM of the number of cells indicated between parentheses; the statistical significance was assessed using the student’s T-test for paired samples: *p < 0.05; **p < 0.01.

Article Snippet: Both the cells and tissue sections were then incubated overnight at 4 °C with the primary antibodies: rabbit anti-P2Y 13 (1:100, Alomone Labs Cat# APR-009), rabbit anti-P2Y 1 (1:100, Alomone Labs Cat# APR-017), rabbit anti-SOX2 (1:100, ABclonal Cat# A0561), mouse anti-GFAP (1:200, Sigma-Aldrich Cat# G3893), mouse anti-βIII-Tubulin (1:800, Sigma-Aldrich Cat# T8660), guinea pig anti-(Doublecortin)DCX (1:400, Millipore Cat# AB2253), mouse anti-ASCL1 (1:100, BD Biosciences Cat# 556604), rabbit anti-KI67 (1:100, Fisher Scientific Cat# RM-9106-S), and chicken anti-GFP (1:400, AvesLab Cat# GFP-1020).

Techniques: Derivative Assay, Concentration Assay, Fluorescence, Immunocytochemistry, Incubation

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: P2Y 13 silencing increases the number of NSCs remaining in the SEZ without promoting proliferation. A. The effect of local P2Y 13 receptor silencing on the NSC population, achieved using the CRISPR/Cas9 sgP2Y 13 lentiviral vectors: Cas9 (Green), SOX2 (Magenta) and GFAP (White), the cell nuclei are stained with DAPI. The lower panels show a higher magnification of the selected areas (scale bar 50 µm). B. Quantification of the Cas9 + /GFAP + /SOX2 + cells remaining in the adult SEZ (n=4). C Effect of the local silencing of the P2Y 13 receptor using the CRISPR/Cas9 sgP2Y 13 lentiviral vectors on the proliferation within the SEZ: Cas9 (Green), Ki67 (Magenta) and GFAP (White), the cell nuclei are stained with DAPI. The lower panels show a higher magnification of the selected areas (scale bar 50 µm). D . Quantification of the Cas9 + /Ki67 + cells SEZ (n=4). E . Effect of local silencing of the P2Y 13 receptor using the CRISPR/Cas9 sgP2Y 13 lentiviral vectors on the neuroblast population: Cas9 (Green), DCX (White) and the cell nuclei are stained with DAPI. The lower panels show the higher magnification of the selected areas (scale bar 50 µm). F. Quantification of the Cas9 + /DCX + cells (n=4). All graphs show mean ±SEM: ***p<0.001 (T-test).

Article Snippet: Both the cells and tissue sections were then incubated overnight at 4 °C with the primary antibodies: rabbit anti-P2Y 13 (1:100, Alomone Labs Cat# APR-009), rabbit anti-P2Y 1 (1:100, Alomone Labs Cat# APR-017), rabbit anti-SOX2 (1:100, ABclonal Cat# A0561), mouse anti-GFAP (1:200, Sigma-Aldrich Cat# G3893), mouse anti-βIII-Tubulin (1:800, Sigma-Aldrich Cat# T8660), guinea pig anti-(Doublecortin)DCX (1:400, Millipore Cat# AB2253), mouse anti-ASCL1 (1:100, BD Biosciences Cat# 556604), rabbit anti-KI67 (1:100, Fisher Scientific Cat# RM-9106-S), and chicken anti-GFP (1:400, AvesLab Cat# GFP-1020).

Techniques: CRISPR, Staining

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: Scheme of the strategy to generate lentiviral vectors for the local overexpression of the P2Y 13 receptor. B . Experimental design of lentiviral injection for local overexpression or silencing of the P2Y 13 receptor. C . Positive control of the local overexpression of P2Y 13 receptor. Lentiviral injection in the adult SEZ demonstrated that all LV-GFP-P2Y 13 transduced cells (Green) co-localized with P2Y 13 expression (red).

Article Snippet: Both the cells and tissue sections were then incubated overnight at 4 °C with the primary antibodies: rabbit anti-P2Y 13 (1:100, Alomone Labs Cat# APR-009), rabbit anti-P2Y 1 (1:100, Alomone Labs Cat# APR-017), rabbit anti-SOX2 (1:100, ABclonal Cat# A0561), mouse anti-GFAP (1:200, Sigma-Aldrich Cat# G3893), mouse anti-βIII-Tubulin (1:800, Sigma-Aldrich Cat# T8660), guinea pig anti-(Doublecortin)DCX (1:400, Millipore Cat# AB2253), mouse anti-ASCL1 (1:100, BD Biosciences Cat# 556604), rabbit anti-KI67 (1:100, Fisher Scientific Cat# RM-9106-S), and chicken anti-GFP (1:400, AvesLab Cat# GFP-1020).

Techniques: Over Expression, Injection, Positive Control, Expressing

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: Local overexpression of P2Y 13 receptors augments the proportion of cells in the RMS and decreases the number of NSCs remaining in the ventral wall of the SEZ. A. Effect of P2Y 13 on SEZ dynamics, with local overexpression promoting more cells entering the RMS and less cells remaining in the SEZ (Scale bar 50 µm). B Quantification of GFP positive cells in the ventral wall of the SEZ and RMS following LV-GFP injection (n=4). C Quantification of GFP + cells in the ventral wall of the SEZ and RMS following LV-P2Y 13 -GFP injections (n=4, scale bar 50 µm). D-E. Effect of local overexpression on the remaining GFAP + (white)/SOX2 + (red)/GFP + (Green) cells in the SEZ after LV-GFP or LV-P2Y 13 -GFP injections (n=4, scale bar 50 µM). F. Quantification of GFAP + /SOX2 + /GFP + cells in the ventral wall of the SEZ following LV-GFP or LV-P2Y 13 -GFP injections (n=4). All graphs show the mean ±SEM: *p<0.05 (T-test).

Article Snippet: Both the cells and tissue sections were then incubated overnight at 4 °C with the primary antibodies: rabbit anti-P2Y 13 (1:100, Alomone Labs Cat# APR-009), rabbit anti-P2Y 1 (1:100, Alomone Labs Cat# APR-017), rabbit anti-SOX2 (1:100, ABclonal Cat# A0561), mouse anti-GFAP (1:200, Sigma-Aldrich Cat# G3893), mouse anti-βIII-Tubulin (1:800, Sigma-Aldrich Cat# T8660), guinea pig anti-(Doublecortin)DCX (1:400, Millipore Cat# AB2253), mouse anti-ASCL1 (1:100, BD Biosciences Cat# 556604), rabbit anti-KI67 (1:100, Fisher Scientific Cat# RM-9106-S), and chicken anti-GFP (1:400, AvesLab Cat# GFP-1020).

Techniques: Over Expression, Injection

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: Transcriptomic analysis of the RNA-seq data obtained from P2Y 13 overexpressing cells in vivo . A. Scheme of the experimental design: right panel created with BioRender.com. B. A principal component analysis (PCA) of the most variably expressed (top 3,000) genes of the samples profiled, colored by condition: control ( FO_C , blue), P2Y 13 overexpression ( FO_Y , green). C. Heat-map of the top 1,000 most variably expressed genes across the samples profiled. The expression of each gene was scaled as a z-score, and the genes and sample labels were sorted by hierarchical clustering. D. Heat-map of the genes associated with the activation/quiescence equilibrium and self-renewal. The expression of each gene was scaled as a z-score, and the genes and sample labels were sorted by hierarchical clustering. E. Volcano plot showing the differential expression of genes between P2Y 13 overexpressing and control samples (n=3, PY 13 and 3 controls). Differentially expressed genes (DEGs, adjusted p-value <0.05) are in red and green when upregulated or downregulated in the P2Y 13 overexpressing samples, respectively. The bar plot represents the number of DEGs. F. Overrepresentation of gene ontology (GO) terms for biological processes from the DEGs (adjusted p-value <0.05) between the P2Y 13 overexpressing and control samples. A customized selection (18) of significant GO terms with the highest gene ratio (top 100) is displayed for clearer representation. The terms are ordered by significance, representing their adjusted p-value (x-axis), and the expressed genes were used as the background in this analysis. G. Stacked bar plot of the inferred cellular composition of neural progenitors for each condition. The cell deconvolutional analysis was carried out using CIBERSORTx, and the gene signatures of the neural progenitors were retrieved from the data generated in Belenguer et al., 2021.

Article Snippet: Both the cells and tissue sections were then incubated overnight at 4 °C with the primary antibodies: rabbit anti-P2Y 13 (1:100, Alomone Labs Cat# APR-009), rabbit anti-P2Y 1 (1:100, Alomone Labs Cat# APR-017), rabbit anti-SOX2 (1:100, ABclonal Cat# A0561), mouse anti-GFAP (1:200, Sigma-Aldrich Cat# G3893), mouse anti-βIII-Tubulin (1:800, Sigma-Aldrich Cat# T8660), guinea pig anti-(Doublecortin)DCX (1:400, Millipore Cat# AB2253), mouse anti-ASCL1 (1:100, BD Biosciences Cat# 556604), rabbit anti-KI67 (1:100, Fisher Scientific Cat# RM-9106-S), and chicken anti-GFP (1:400, AvesLab Cat# GFP-1020).

Techniques: RNA Sequencing Assay, In Vivo, Control, Over Expression, Expressing, Activation Assay, Selection, Generated

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: Neurogenic trees from the ventral wall of the SEZ tracked in culture following treatment with the P2Y 13 agonist 2MesADP, in the presence or absence of the MRS2211 antagonist. A Representative symmetric neurogenic tree obtained in control conditions after 6 days in culture (N, neuron; X, cell death). B Complex symmetric neurogenic trees of 5 rounds of division obtained in the presence of 2MeSADP after 6 days in culture (N, neuron; X, cell death). The phase contrast images in both A and B depict the lineage progression in the live imaging experiment (day-hour-min). The last images show post-imaging immunocytochemistry of the neuroblast progeny (βIII-tubulin in green) Scale bar 30 µm. C Clones undergoing 1-6 rounds of division in the live imaging experiments (n=5) (n=4 for MRS2211). D Summary of all the clones tracked undergoing 4 or 5 rounds of division in our live imaging experiments, either in control conditions, or when exposed to 2MeSADP or 2MeSADP + MRS2211 (n=5) (n=4 for MRS2211). E Cell survival in the lineage trees (n=5). In all cases the progeny generated were identified by post-imaging immunocytochemistry. All graphs show the mean ±SEM: *p<0.05, and **p<0.01 (ANOVA with a Tukey’s post-test).

Article Snippet: Both the cells and tissue sections were then incubated overnight at 4 °C with the primary antibodies: rabbit anti-P2Y 13 (1:100, Alomone Labs Cat# APR-009), rabbit anti-P2Y 1 (1:100, Alomone Labs Cat# APR-017), rabbit anti-SOX2 (1:100, ABclonal Cat# A0561), mouse anti-GFAP (1:200, Sigma-Aldrich Cat# G3893), mouse anti-βIII-Tubulin (1:800, Sigma-Aldrich Cat# T8660), guinea pig anti-(Doublecortin)DCX (1:400, Millipore Cat# AB2253), mouse anti-ASCL1 (1:100, BD Biosciences Cat# 556604), rabbit anti-KI67 (1:100, Fisher Scientific Cat# RM-9106-S), and chicken anti-GFP (1:400, AvesLab Cat# GFP-1020).

Techniques: Control, Imaging, Immunocytochemistry, Clone Assay, Generated

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: Effect of the P2Y 13 receptor on NSC self-renewal. A . Quantification of the symmetric lineage trees (generating only neuroblasts) and the asymmetric lineage trees (generating neuroblasts and new NSCs through self-renewal events) in control conditions, or on exposure to 2MeSADP or 2MeSADP + MRS2211 (n=4). B. Proportion of GFAP + /SOX2 + NSCs relative to the total number of cells in the culture according to the following labels: “Initial” indicates the proportion of GFAP + /SOX2 + in the culture and the “Quiescent” cells are the fraction of the initial cells that remain quiescent throughout the live imaging experiments. The “Clones” reflect the fraction of the initial cells that undergo lineage progression (Control and 2MeSADP n=5 and 2MeSADP+MRS2211 n = 4). C. The models represent the stereotypic behavior of NSCs under control conditions and when exposed to 2MeSADP or 2MeSADP+MRS211. Note how 2MeSADP increases the speed in the cell cycle while impeding self-renewal divisions. By contrast, MRS2211 instructs NSCs to remain quiescent. D. Cell Cycle length within the complex trees tracked (≥4 rounds of division). E . Cell Cycle length for the first division within the complex trees tracked (≥4 rounds of division, n= 5). Note that this first division is normally associated with slow-dividing astroglia, yet 2MeSADP significantly increase the speed of the cell cycle (n=5). All the graphs show the mean ±SEM: *p<0.05, **p<0.01 (T-test for D, E and ANOVA with a Tukeys post-test for B ).

Article Snippet: Both the cells and tissue sections were then incubated overnight at 4 °C with the primary antibodies: rabbit anti-P2Y 13 (1:100, Alomone Labs Cat# APR-009), rabbit anti-P2Y 1 (1:100, Alomone Labs Cat# APR-017), rabbit anti-SOX2 (1:100, ABclonal Cat# A0561), mouse anti-GFAP (1:200, Sigma-Aldrich Cat# G3893), mouse anti-βIII-Tubulin (1:800, Sigma-Aldrich Cat# T8660), guinea pig anti-(Doublecortin)DCX (1:400, Millipore Cat# AB2253), mouse anti-ASCL1 (1:100, BD Biosciences Cat# 556604), rabbit anti-KI67 (1:100, Fisher Scientific Cat# RM-9106-S), and chicken anti-GFP (1:400, AvesLab Cat# GFP-1020).

Techniques: Control, Imaging, Clone Assay

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Changes of C3d/S100A10 levels, and pan-reactive and A1/A2 specific gene expression in reactive astrocytes after treatment with microglia-conditioned media (MCM) and P2Y 1 R-ANT. (a), (b). Representative images (a) and quantitative data (b) of western blots showing protein levels of GFAP, C3d, S100A10, and CSPG in non-OGD astrocytes, OGD astrocytes, OGD astrocytes treated with MCM, OGD astrocytes treated with P2Y 1 R-ANT, and OGD astrocytes treated with P2Y 1 R-ANT and MCM. β-actin was used as an internal control. * P < 0.05 vs. non-OGD astrocyte, # P < 0.05 vs. OGD astrocytes. (c). Venn diagram of mRNAs with upregulated (fold change ≥ 1.5) and downregulated (fold change ≤ 0.67) expressions in OGD astrocytes, OGD astrocytes treated with P2Y 1 R-ANT (1 mM), and OGD astrocytes treated with P2Y 1 R-ANT (1 mM) and MCM. (d). Heatmap of the entire mRNA expression in OGD astrocytes, OGD astrocytes treated with P2Y 1 R-ANT (1 mM), and OGD astrocytes treated with P2Y 1 R-ANT (1 mM) and MCM. (e). Heatmaps comparing the mean expression of pan-reactive, A1-specific, and A2 specific genes in OGD astrocytes; OGD astrocytes treated with P2Y 1 R-ANT (1 mM); and OGD astrocytes treated with P2Y 1 R-ANT (1 mM) and MCM. N = 4/group. Values are the mean ± SD. GFAP = glial fibrillary acidic protein, CSPG = chondroitin sulfate proteoglycans, OGD = oxygen–glucose deprivation, P2Y 1 R-ANT = P2Y 1 receptor antagonist, MCM = microglial conditioned medium

Article Snippet: The primary antibodies used in this study were rabbit polyclonal anti-ionized calcium-binding adapter molecule 1 (Iba-1) (1:800; Wako), mouse monoclonal anti-glial fibrillary acidic protein (GFAP) (1:200; MBL), goat polyclonal anti-GFAP (1:200; Abcam), goat polyclonal anti-C3d (1:25, R&D Systems), rabbit polyclonal anti-S100A10 (1:50, Proteintech), rabbit polyclonal anti-P2Y 1 receptor antibody (1:100, Alomone Labs), rabbit polyclonal anti-nuclear factor-κβ (NF-κB) (1:200; Abcam), mouse monoclonal anti-TNF-α (1:200; GeneTex), mouse monoclonal pNFH (SMI31)(1:200; BioLegend), and mouse MAP2 (1:500; Merck).

Techniques: Expressing, Western Blot, Control

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Change in inflammatory gene expression and pathway analysis. (a). Top 20 significant canonical pathways of the core analysis in IPA of most highly expressed genes in OGD astrocytes treated with P2Y 1 R-ANT (1 mM) and MCM relative to OGD astrocytes. Blue bars: negative z-score; orange bars: positive z-score; gray bars: no activity pattern available; white bars: activity of zero. (b), (c). Heatmap of mRNA-related Neuroinflammation Signaling expression, and quantitative analysis of representative mRNA-related Neuroinflammation Signaling in OGD astrocytes, OGD astrocytes treated with P2Y 1 R-ANT (1 mM), and OGD astrocytes treated with P2Y 1 R-ANT (1 mM) and MCM. * P < 0.05 vs. OGD astrocyte, # P < 0.05 vs. OGD astrocytes treated with P2Y 1 R-ANT. (d). Signaling pathway predicted by analyzing changes using IPA software in mRNA expression in the OGD astrocytes treated with P2Y 1 R-ANT and MCM relative to OGD astrocytes, or OGD astrocytes treated with P2Y 1 R-ANT. The functional networks were generated via IPA (QIAGEN Inc., https://www.qiagenbio-informatics.com/products/ingenuity-pathway-analysis ). N = 4/group. Values are the mean ± SD. IPA = Ingenuity Pathway Analysis, P2Y 1 R-ANT = P2Y 1 receptor antagonist, MCM = microglial conditioned medium, MAPK = mitogen-activated protein kinase, NF-κB = nuclear factor-κβ, TNF-α = tumor necrosis factor, IL-1β = interleukin-1β, NOX = nitrogen oxides, CASP8 = Caspase 8

Article Snippet: The primary antibodies used in this study were rabbit polyclonal anti-ionized calcium-binding adapter molecule 1 (Iba-1) (1:800; Wako), mouse monoclonal anti-glial fibrillary acidic protein (GFAP) (1:200; MBL), goat polyclonal anti-GFAP (1:200; Abcam), goat polyclonal anti-C3d (1:25, R&D Systems), rabbit polyclonal anti-S100A10 (1:50, Proteintech), rabbit polyclonal anti-P2Y 1 receptor antibody (1:100, Alomone Labs), rabbit polyclonal anti-nuclear factor-κβ (NF-κB) (1:200; Abcam), mouse monoclonal anti-TNF-α (1:200; GeneTex), mouse monoclonal pNFH (SMI31)(1:200; BioLegend), and mouse MAP2 (1:500; Merck).

Techniques: Expressing, Activity Assay, Software, Functional Assay, Generated

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Significance of AEVs derived from anti-inflammatory astrocytes to the peri-infarct area after MCAO. (a). The experimental scheme of isolating AEVs from OGD astrocytes treated with MCM and P2Y 1 R-ANT and their application for rats subjected to MCAO and cultured cortical neurons. (b), (c). Modified neurological severity score ( b ) and latency to fall off the rotarod at 56 days after MCAO ( c ) in PBS treatment, treatment with AEVs derived from OGD astrocytes (100 µg), and OGD astrocytes treated with MCM and P2Y 1 R-ANT (100 µg) in rats subjected to MCAO. N = 6–7/group. Values are the mean ± SD. (d), (e). Double immunofluorescent confocal images and quantitative data of the peri-infarct area at 56 days after MCAO with intracerebral administration of PBS, 100 µg AEVs derived from OGD astrocytes, and 100 µg AEVs derived from OGD astrocytes treated with MCM and P2Y 1 R-ANT, showing C3d + area (green) ( d ) and S100A10 + area (green) ( e ), and co-localized with GFAP + area (yellow). Merge ratio of C3d /GFAP ( d ), and S100A10/GFAP ( e ). N = 5/group (three sections per rat, and total of 15 samples in each group). Values are the mean ± SD. * P < 0.05 vs. PBS-treated rats, # P < 0.05 vs. rats treated with AEVs derived from OGD astrocytes. Scale bar = 100 μm AEVs = astrocytic extracellular vesicles, MCAO = middle cerebral artery occlusion, P2Y 1 R-ANT = P2Y 1 receptor antagonist, MCM = microglia-conditioned medium, GFAP = glial fibrillary acidic protein, OGD = oxygen–glucose deprivation

Article Snippet: The primary antibodies used in this study were rabbit polyclonal anti-ionized calcium-binding adapter molecule 1 (Iba-1) (1:800; Wako), mouse monoclonal anti-glial fibrillary acidic protein (GFAP) (1:200; MBL), goat polyclonal anti-GFAP (1:200; Abcam), goat polyclonal anti-C3d (1:25, R&D Systems), rabbit polyclonal anti-S100A10 (1:50, Proteintech), rabbit polyclonal anti-P2Y 1 receptor antibody (1:100, Alomone Labs), rabbit polyclonal anti-nuclear factor-κβ (NF-κB) (1:200; Abcam), mouse monoclonal anti-TNF-α (1:200; GeneTex), mouse monoclonal pNFH (SMI31)(1:200; BioLegend), and mouse MAP2 (1:500; Merck).

Techniques: Derivative Assay, Cell Culture, Modification

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Expression of microRNAs (miRNAs) in AEVs and inflammatory regulation in peri-infarct glial scars. (a). Heatmap of miRNA profiles on AEVs derived from OGD astrocytes and OGD astrocytes treated with P2Y 1 R-ANT and MCM. (b). Quantitative analysis of representative miRNAs related to ‘Inflammatory Response’ in AEVs derived from OGD astrocytes treated with P2Y 1 R-ANT and MCM, relative to AEVs derived from OGD astrocytes. N = 4/group. Values are the mean ± SD. * P < 0.05 vs. AEVs derived from OGD astrocytes. (c), (d). Double immunofluorescent confocal images and quantitative data of the peri-infarct area 56 days after MCAO with intracerebral administration of PBS, 100 µg AEVs derived from OGD astrocytes, and 100 µg AEVs derived from OGD astrocytes treated with MCM and P2Y 1 R-ANT, showing TNFα + area (green) ( c ) and NF-κB + area (green) ( d ), and co-localized with GFAP + area (yellow). N = 5/group (three sections per rat, and total of 15 samples in each group). Values are the mean ± SD. * P < 0.05 vs. PBS-treated rats. Scale bar = 100 μm AEVs = astrocytic extracellular vesicles, P2Y 1 R-ANT = P2Y 1 receptor antagonist

Article Snippet: The primary antibodies used in this study were rabbit polyclonal anti-ionized calcium-binding adapter molecule 1 (Iba-1) (1:800; Wako), mouse monoclonal anti-glial fibrillary acidic protein (GFAP) (1:200; MBL), goat polyclonal anti-GFAP (1:200; Abcam), goat polyclonal anti-C3d (1:25, R&D Systems), rabbit polyclonal anti-S100A10 (1:50, Proteintech), rabbit polyclonal anti-P2Y 1 receptor antibody (1:100, Alomone Labs), rabbit polyclonal anti-nuclear factor-κβ (NF-κB) (1:200; Abcam), mouse monoclonal anti-TNF-α (1:200; GeneTex), mouse monoclonal pNFH (SMI31)(1:200; BioLegend), and mouse MAP2 (1:500; Merck).

Techniques: Expressing, Derivative Assay

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Axonal outgrowth after AEV treatment and hindering by TNF-α (a), (b). Double immunofluorescent confocal images and quantitative data of the peri-infarct area 56 days after MCAO with intracerebral administration of PBS, 100 µg AEVs derived from OGD astrocytes, and 100 µg AEVs derived from OGD astrocytes treated with MCM and P2Y 1 R-ANT, showing pNFH + axons (green) ( a ) and MAP2 cells + (green) ( b ), with GFAP. N = 5/group (three sections per rat, and total of 15 samples in each group). Values are the mean ± SD. Scale bar = 100 μm. (c). Representative time-lapse microscopic images and quantitative data of primary cortical neurons in a microfluidic chamber showing axonal elongation (distance from yellow arrow to red arrow) in OGD neurons, OGD neurons treated with 1 ng/µl of TNF-α, and OGD neurons treated with 10 ng/µl of TNF-α. N = 3/group. Values are the mean ± SD. Scale bar = 20 μm. Quantitative data of axonal elongation per 30 min prior to 96 h after OGD. * P < 0.05, OGD neurons treated with 1 ng/µL of TNF-α vs. OGD neurons; # P < 0.05, OGD neurons treated with 10 ng/µL of TNF-α vs. OGD neurons; $ P < 0.05, OGD neurons treated with 10 ng/µL of TNF-α vs. OGD neurons treated with 1 ng/µL of TNF-α. AEVs = astrocytic extracellular vesicles, MCAO = middle cerebral artery occlusion, OGD = oxygen–glucose deprivation, P2Y 1 R-ANT = P2Y 1 receptor antagonist, MCM = microglia-conditioned medium, GFAP = glial fibrillary acidic protein, pNFH = phosphorylated neurofilament heavy chain

Article Snippet: The primary antibodies used in this study were rabbit polyclonal anti-ionized calcium-binding adapter molecule 1 (Iba-1) (1:800; Wako), mouse monoclonal anti-glial fibrillary acidic protein (GFAP) (1:200; MBL), goat polyclonal anti-GFAP (1:200; Abcam), goat polyclonal anti-C3d (1:25, R&D Systems), rabbit polyclonal anti-S100A10 (1:50, Proteintech), rabbit polyclonal anti-P2Y 1 receptor antibody (1:100, Alomone Labs), rabbit polyclonal anti-nuclear factor-κβ (NF-κB) (1:200; Abcam), mouse monoclonal anti-TNF-α (1:200; GeneTex), mouse monoclonal pNFH (SMI31)(1:200; BioLegend), and mouse MAP2 (1:500; Merck).

Techniques: Derivative Assay

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Diagram depicting the findings of the present study. Microglia and inhibition of P2Y 1 R regulate reactive astrocytes by transforming C3d/S100A10 expression and suppressing neuroinflammation. AEVs derived from reactive astrocytes with anti-inflammatory properties possessing miR-146a-5p regulate glial scars by suppressing NF-κB and TNF-α, which is permissive for axonal outgrowth and improves stroke recovery

Article Snippet: The primary antibodies used in this study were rabbit polyclonal anti-ionized calcium-binding adapter molecule 1 (Iba-1) (1:800; Wako), mouse monoclonal anti-glial fibrillary acidic protein (GFAP) (1:200; MBL), goat polyclonal anti-GFAP (1:200; Abcam), goat polyclonal anti-C3d (1:25, R&D Systems), rabbit polyclonal anti-S100A10 (1:50, Proteintech), rabbit polyclonal anti-P2Y 1 receptor antibody (1:100, Alomone Labs), rabbit polyclonal anti-nuclear factor-κβ (NF-κB) (1:200; Abcam), mouse monoclonal anti-TNF-α (1:200; GeneTex), mouse monoclonal pNFH (SMI31)(1:200; BioLegend), and mouse MAP2 (1:500; Merck).

Techniques: Inhibition, Expressing, Derivative Assay

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Changes of C3d/S100A10 levels, and pan-reactive and A1/A2 specific gene expression in reactive astrocytes after treatment with microglia-conditioned media (MCM) and P2Y 1 R-ANT. (a), (b). Representative images (a) and quantitative data (b) of western blots showing protein levels of GFAP, C3d, S100A10, and CSPG in non-OGD astrocytes, OGD astrocytes, OGD astrocytes treated with MCM, OGD astrocytes treated with P2Y 1 R-ANT, and OGD astrocytes treated with P2Y 1 R-ANT and MCM. β-actin was used as an internal control. * P < 0.05 vs. non-OGD astrocyte, # P < 0.05 vs. OGD astrocytes. (c). Venn diagram of mRNAs with upregulated (fold change ≥ 1.5) and downregulated (fold change ≤ 0.67) expressions in OGD astrocytes, OGD astrocytes treated with P2Y 1 R-ANT (1 mM), and OGD astrocytes treated with P2Y 1 R-ANT (1 mM) and MCM. (d). Heatmap of the entire mRNA expression in OGD astrocytes, OGD astrocytes treated with P2Y 1 R-ANT (1 mM), and OGD astrocytes treated with P2Y 1 R-ANT (1 mM) and MCM. (e). Heatmaps comparing the mean expression of pan-reactive, A1-specific, and A2 specific genes in OGD astrocytes; OGD astrocytes treated with P2Y 1 R-ANT (1 mM); and OGD astrocytes treated with P2Y 1 R-ANT (1 mM) and MCM. N = 4/group. Values are the mean ± SD. GFAP = glial fibrillary acidic protein, CSPG = chondroitin sulfate proteoglycans, OGD = oxygen–glucose deprivation, P2Y 1 R-ANT = P2Y 1 receptor antagonist, MCM = microglial conditioned medium

Article Snippet: The primary antibodies used in this study were goat polyclonal anti-GFAP (1:40,000; Abcam), goat polyclonal anti-C3d (a marker for A1 astrocytes) (1:2,000, R&D Systems), rabbit polyclonal anti-S100A10 (1:2,000, Proteintech), mouse monoclonal anti-CSPG (1:100,000, Abcam), mouse monoclonal anti-phosphorylated neurofilament heavy chain (pNFH) (1:500; BioLegend), rabbit polyclonal anti-P2Y 1 R-ANT (1:200; Alomone Labs), rabbit polyclonal anti-caspase-3 (1:500, Abcam), and rabbit monoclonal anti-actin (1:10,000, Abcam).

Techniques: Expressing, Western Blot, Control

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Change in inflammatory gene expression and pathway analysis. (a). Top 20 significant canonical pathways of the core analysis in IPA of most highly expressed genes in OGD astrocytes treated with P2Y 1 R-ANT (1 mM) and MCM relative to OGD astrocytes. Blue bars: negative z-score; orange bars: positive z-score; gray bars: no activity pattern available; white bars: activity of zero. (b), (c). Heatmap of mRNA-related Neuroinflammation Signaling expression, and quantitative analysis of representative mRNA-related Neuroinflammation Signaling in OGD astrocytes, OGD astrocytes treated with P2Y 1 R-ANT (1 mM), and OGD astrocytes treated with P2Y 1 R-ANT (1 mM) and MCM. * P < 0.05 vs. OGD astrocyte, # P < 0.05 vs. OGD astrocytes treated with P2Y 1 R-ANT. (d). Signaling pathway predicted by analyzing changes using IPA software in mRNA expression in the OGD astrocytes treated with P2Y 1 R-ANT and MCM relative to OGD astrocytes, or OGD astrocytes treated with P2Y 1 R-ANT. The functional networks were generated via IPA (QIAGEN Inc., https://www.qiagenbio-informatics.com/products/ingenuity-pathway-analysis ). N = 4/group. Values are the mean ± SD. IPA = Ingenuity Pathway Analysis, P2Y 1 R-ANT = P2Y 1 receptor antagonist, MCM = microglial conditioned medium, MAPK = mitogen-activated protein kinase, NF-κB = nuclear factor-κβ, TNF-α = tumor necrosis factor, IL-1β = interleukin-1β, NOX = nitrogen oxides, CASP8 = Caspase 8

Article Snippet: The primary antibodies used in this study were goat polyclonal anti-GFAP (1:40,000; Abcam), goat polyclonal anti-C3d (a marker for A1 astrocytes) (1:2,000, R&D Systems), rabbit polyclonal anti-S100A10 (1:2,000, Proteintech), mouse monoclonal anti-CSPG (1:100,000, Abcam), mouse monoclonal anti-phosphorylated neurofilament heavy chain (pNFH) (1:500; BioLegend), rabbit polyclonal anti-P2Y 1 R-ANT (1:200; Alomone Labs), rabbit polyclonal anti-caspase-3 (1:500, Abcam), and rabbit monoclonal anti-actin (1:10,000, Abcam).

Techniques: Expressing, Activity Assay, Software, Functional Assay, Generated

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Significance of AEVs derived from anti-inflammatory astrocytes to the peri-infarct area after MCAO. (a). The experimental scheme of isolating AEVs from OGD astrocytes treated with MCM and P2Y 1 R-ANT and their application for rats subjected to MCAO and cultured cortical neurons. (b), (c). Modified neurological severity score ( b ) and latency to fall off the rotarod at 56 days after MCAO ( c ) in PBS treatment, treatment with AEVs derived from OGD astrocytes (100 µg), and OGD astrocytes treated with MCM and P2Y 1 R-ANT (100 µg) in rats subjected to MCAO. N = 6–7/group. Values are the mean ± SD. (d), (e). Double immunofluorescent confocal images and quantitative data of the peri-infarct area at 56 days after MCAO with intracerebral administration of PBS, 100 µg AEVs derived from OGD astrocytes, and 100 µg AEVs derived from OGD astrocytes treated with MCM and P2Y 1 R-ANT, showing C3d + area (green) ( d ) and S100A10 + area (green) ( e ), and co-localized with GFAP + area (yellow). Merge ratio of C3d /GFAP ( d ), and S100A10/GFAP ( e ). N = 5/group (three sections per rat, and total of 15 samples in each group). Values are the mean ± SD. * P < 0.05 vs. PBS-treated rats, # P < 0.05 vs. rats treated with AEVs derived from OGD astrocytes. Scale bar = 100 μm AEVs = astrocytic extracellular vesicles, MCAO = middle cerebral artery occlusion, P2Y 1 R-ANT = P2Y 1 receptor antagonist, MCM = microglia-conditioned medium, GFAP = glial fibrillary acidic protein, OGD = oxygen–glucose deprivation

Article Snippet: The primary antibodies used in this study were goat polyclonal anti-GFAP (1:40,000; Abcam), goat polyclonal anti-C3d (a marker for A1 astrocytes) (1:2,000, R&D Systems), rabbit polyclonal anti-S100A10 (1:2,000, Proteintech), mouse monoclonal anti-CSPG (1:100,000, Abcam), mouse monoclonal anti-phosphorylated neurofilament heavy chain (pNFH) (1:500; BioLegend), rabbit polyclonal anti-P2Y 1 R-ANT (1:200; Alomone Labs), rabbit polyclonal anti-caspase-3 (1:500, Abcam), and rabbit monoclonal anti-actin (1:10,000, Abcam).

Techniques: Derivative Assay, Cell Culture, Modification

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Expression of microRNAs (miRNAs) in AEVs and inflammatory regulation in peri-infarct glial scars. (a). Heatmap of miRNA profiles on AEVs derived from OGD astrocytes and OGD astrocytes treated with P2Y 1 R-ANT and MCM. (b). Quantitative analysis of representative miRNAs related to ‘Inflammatory Response’ in AEVs derived from OGD astrocytes treated with P2Y 1 R-ANT and MCM, relative to AEVs derived from OGD astrocytes. N = 4/group. Values are the mean ± SD. * P < 0.05 vs. AEVs derived from OGD astrocytes. (c), (d). Double immunofluorescent confocal images and quantitative data of the peri-infarct area 56 days after MCAO with intracerebral administration of PBS, 100 µg AEVs derived from OGD astrocytes, and 100 µg AEVs derived from OGD astrocytes treated with MCM and P2Y 1 R-ANT, showing TNFα + area (green) ( c ) and NF-κB + area (green) ( d ), and co-localized with GFAP + area (yellow). N = 5/group (three sections per rat, and total of 15 samples in each group). Values are the mean ± SD. * P < 0.05 vs. PBS-treated rats. Scale bar = 100 μm AEVs = astrocytic extracellular vesicles, P2Y 1 R-ANT = P2Y 1 receptor antagonist

Article Snippet: The primary antibodies used in this study were goat polyclonal anti-GFAP (1:40,000; Abcam), goat polyclonal anti-C3d (a marker for A1 astrocytes) (1:2,000, R&D Systems), rabbit polyclonal anti-S100A10 (1:2,000, Proteintech), mouse monoclonal anti-CSPG (1:100,000, Abcam), mouse monoclonal anti-phosphorylated neurofilament heavy chain (pNFH) (1:500; BioLegend), rabbit polyclonal anti-P2Y 1 R-ANT (1:200; Alomone Labs), rabbit polyclonal anti-caspase-3 (1:500, Abcam), and rabbit monoclonal anti-actin (1:10,000, Abcam).

Techniques: Expressing, Derivative Assay

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Axonal outgrowth after AEV treatment and hindering by TNF-α (a), (b). Double immunofluorescent confocal images and quantitative data of the peri-infarct area 56 days after MCAO with intracerebral administration of PBS, 100 µg AEVs derived from OGD astrocytes, and 100 µg AEVs derived from OGD astrocytes treated with MCM and P2Y 1 R-ANT, showing pNFH + axons (green) ( a ) and MAP2 cells + (green) ( b ), with GFAP. N = 5/group (three sections per rat, and total of 15 samples in each group). Values are the mean ± SD. Scale bar = 100 μm. (c). Representative time-lapse microscopic images and quantitative data of primary cortical neurons in a microfluidic chamber showing axonal elongation (distance from yellow arrow to red arrow) in OGD neurons, OGD neurons treated with 1 ng/µl of TNF-α, and OGD neurons treated with 10 ng/µl of TNF-α. N = 3/group. Values are the mean ± SD. Scale bar = 20 μm. Quantitative data of axonal elongation per 30 min prior to 96 h after OGD. * P < 0.05, OGD neurons treated with 1 ng/µL of TNF-α vs. OGD neurons; # P < 0.05, OGD neurons treated with 10 ng/µL of TNF-α vs. OGD neurons; $ P < 0.05, OGD neurons treated with 10 ng/µL of TNF-α vs. OGD neurons treated with 1 ng/µL of TNF-α. AEVs = astrocytic extracellular vesicles, MCAO = middle cerebral artery occlusion, OGD = oxygen–glucose deprivation, P2Y 1 R-ANT = P2Y 1 receptor antagonist, MCM = microglia-conditioned medium, GFAP = glial fibrillary acidic protein, pNFH = phosphorylated neurofilament heavy chain

Article Snippet: The primary antibodies used in this study were goat polyclonal anti-GFAP (1:40,000; Abcam), goat polyclonal anti-C3d (a marker for A1 astrocytes) (1:2,000, R&D Systems), rabbit polyclonal anti-S100A10 (1:2,000, Proteintech), mouse monoclonal anti-CSPG (1:100,000, Abcam), mouse monoclonal anti-phosphorylated neurofilament heavy chain (pNFH) (1:500; BioLegend), rabbit polyclonal anti-P2Y 1 R-ANT (1:200; Alomone Labs), rabbit polyclonal anti-caspase-3 (1:500, Abcam), and rabbit monoclonal anti-actin (1:10,000, Abcam).

Techniques: Derivative Assay

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Diagram depicting the findings of the present study. Microglia and inhibition of P2Y 1 R regulate reactive astrocytes by transforming C3d/S100A10 expression and suppressing neuroinflammation. AEVs derived from reactive astrocytes with anti-inflammatory properties possessing miR-146a-5p regulate glial scars by suppressing NF-κB and TNF-α, which is permissive for axonal outgrowth and improves stroke recovery

Article Snippet: The primary antibodies used in this study were goat polyclonal anti-GFAP (1:40,000; Abcam), goat polyclonal anti-C3d (a marker for A1 astrocytes) (1:2,000, R&D Systems), rabbit polyclonal anti-S100A10 (1:2,000, Proteintech), mouse monoclonal anti-CSPG (1:100,000, Abcam), mouse monoclonal anti-phosphorylated neurofilament heavy chain (pNFH) (1:500; BioLegend), rabbit polyclonal anti-P2Y 1 R-ANT (1:200; Alomone Labs), rabbit polyclonal anti-caspase-3 (1:500, Abcam), and rabbit monoclonal anti-actin (1:10,000, Abcam).

Techniques: Inhibition, Expressing, Derivative Assay

Journal: Molecular Pain

Article Title: Satellite glial cell P2Y 12 receptor in the trigeminal ganglion is involved in lingual neuropathic pain mechanisms in rats

doi: 10.1186/1744-8069-8-23

Figure Lengend Snippet: Expression of P2Y 12 R and GFAP in TG and effect of P2Y 12 R antagonist on GFAP expression in LNC-rats . Photomicrographs of P2Y 12 R-IR cells (A and D); GFAP-IR cells (B); P2Y 12 R-IR and GFAP-IR cells (C); NeuN-IR cells (E); P2Y 12 R-IR and NeuN-IR cells (F) in V3 branch region on day 3 after LNC. Photomicrographs of P2Y 12 R-IR cells (G); GFAP-IR cells (H) in V3 branch region on day 3 after sham operation. Photomicrographs of GFAP-IR cells following MRS2395 administration for 3 successive days into TG in LNC-rats (I). J and K: Size-frequency histograms illustrating distribution of somata of TG neurons encircled with GFAP-IR cells in V3 branch region on day 3 after operation and following daily successive MRS2395 (18.0 ng/day) or vehicle administration (from day 0 to day 2) into TG in LNC-rats (J) and sham-rats (K) on day 3 after LNC or sham operation. Arrows indicate GFAP-IR cells expressing P2Y 12 R-IR cells. *: p < 0.05, **: p < 0.01, ***: p < 0.001 (n = 5 in each group, Student's t -test). Scale bars = 50 μm.

Article Snippet: Sections were incubated with rabbit anti-P2Y 12 R polyclonal antibody (1:200; Anaspec, Fremont, CA), rabbit anti-P2Y 1 R polyclonal antibody (1:300; Alomone labs, Jerusalem, Israel), rabbit anti-P2Y 13 R polyclonal antibody (1:300; Chemicon, Temicula, CA), mouse anti-GFAP monoclonal antibody (1:800; Millipore) and/or mouse anti-NeuN monoclonal antibody (1:1000; Chemicon) in 0.01 M PBS containing 4% NGS and 0.3% Triton X-100 (Sigma-Aldrich) overnight at 4°C.

Techniques: Expressing

Journal: Molecular Pain

Article Title: Satellite glial cell P2Y 12 receptor in the trigeminal ganglion is involved in lingual neuropathic pain mechanisms in rats

doi: 10.1186/1744-8069-8-23

Figure Lengend Snippet: Effect of P2Y 12 R antagonist on nocifensive reflex in LNC-rats . Effect of daily successive administration (from day 0 to day 2) of MRS2395 or vehicle into TG on mean head-withdrawal reflex thresholds to mechanical (A) and heat (B) stimulation to the tongue in the LNC- or sham-rats on day 3 after operation. Head-withdrawal reflex threshold after LNC or sham operation with MRS2395 administration was compared with that before LNC or sham operation with MRS2395 administration. **: p < 0.01, ***: p < 0.001 (n = 6 in each group, Student's t -test).

Article Snippet: Sections were incubated with rabbit anti-P2Y 12 R polyclonal antibody (1:200; Anaspec, Fremont, CA), rabbit anti-P2Y 1 R polyclonal antibody (1:300; Alomone labs, Jerusalem, Israel), rabbit anti-P2Y 13 R polyclonal antibody (1:300; Chemicon, Temicula, CA), mouse anti-GFAP monoclonal antibody (1:800; Millipore) and/or mouse anti-NeuN monoclonal antibody (1:1000; Chemicon) in 0.01 M PBS containing 4% NGS and 0.3% Triton X-100 (Sigma-Aldrich) overnight at 4°C.

Techniques:

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Neuroprotective effects of microglial P2Y 1 receptors against ischemic neuronal injury

doi: 10.1177/0271678X18805317

Figure Lengend Snippet: Physiological parameters and regional cerebral blood flow.

Article Snippet: Isolated microglia were re-suspended in fluorescence-activated cell sorting (FACS) buffer (PBS supplemented with 10% fetal bovine serum) and stained with rabbit anti-P2Y 1 R antibodies (1:200, Alomone Labs, Jerusalem, Israel) for 2 h at 4℃.

Techniques: