Journal: The Journal of pathology

Article Title: Macrophages producing chondroitin sulfate proteoglycan-4 induce neuro-cardiac junction impairment in Duchenne muscular dystrophy.

doi: 10.1002/path.6362

Figure Lengend Snippet: Figure 1. Cardiac fibrosis and innervation. (A) Masson’s trichrome staining of young (3-month-old) and old (10-month-old) WT and mdx mouse hearts. Scale bars, 100 μm. The graph shows the area as a percentage of the fibrosis index (fibrotic area/whole area). N = 3 biological replicates. (B) qPCR for fibrosis-related transcripts in 3- and 10-month-old mdx and WT hearts (Col1a1, Col3a1, Fn1, Tgfb1, Twist 1, Twist 2). N = 3 biological replicates. (C) Confocal images for tyrosine hydroxylase (TH), Synapsin 1 (SYN1), MPs (F4/80), proteoglycan CSPG4, and fibronectin 1 (FN1) on myocardium sections of 3- and 10-month-old mice. The markers TH, SYN1, and CSPG4 are labeled in yellow, cardiac troponin (cTNNT) and F4/80 are in magenta, while FN1 is in white. Scale bars, 10 μm. Nuclei are counterstained with DAPI in blue. (D) Charts indicate positive area expressed as percentage of TH, SYN1, and F4/80 on whole area. N = 3 sections per N = 3 biological replicates. (E and F) Western blot analysis of TH, SYN1, and CSPG4 in mdx and WT hearts of young and old mice. Graphs show optical density (OD) of protein bands normalized to vinculin (VCL). N = 3 biological replicates. Error bars show SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 were calculated using Student’s t-test.

Article Snippet: Western blot Proteins were extracted from tissues using lysis buffer (50 mM TRIS HCl pH 7.5), 0.6 M sucrose (Catalogue No. S0389, Sigma-Aldrich), 50% glycerol (Catalogue No. G5516, Sigma-Aldrich), 1% TRITON, 50 mM NaCl, 10 mM NaF, 2 mM sodium orthovanadate (Na3VO4, Catalogue No. 450243 Sigma-Aldrich), 1 mM PMSF (Catalogue No. 36978, Thermo Scientific,Waltham,MA, USA), 5 mM β-glycerophosphate (Catalogue No. G9422, Sigma-Aldrich), 1000X protease inhibitors (Catalogue No. 04693116001, Roche) for 30 min on ice, sonicated, and centrifuged at 12,000 g for 15 min at 4 C. Proteins were resolved using SDS-PAGE, transferred to nitrocellulose membranes (Catalogue No. GE10600079, Amersham Protran, Johns Creek, GA, USA), blocked with 5% nonfat dry milk powder (Catalogue No. 1.15363, Merck Millipore, Billerica, MA, USA) in PBS 1 for 45 min, and incubated at 4 C overnight with primary antibodies: tyrosine hydroxylase (1:1000, ab152), Synapsin 1 (1:1000, Cell Signaling Technology, #5297), CSPG4 (1:1000, Cell Signaling Technology, #52635), and Vinculin (1:500, V9131, Sigma-Aldrich), followed by incubation with secondary antibodies (antiRabbit NA934AV, 1:5000; anti-Mouse LNXa931/AE, 1:10000, GE Healthcare Life Sciences, Havelock, NE, USA) for 1 h at RT.

Techniques: Staining, Labeling, Western Blot

Journal: The Journal of pathology

Article Title: Macrophages producing chondroitin sulfate proteoglycan-4 induce neuro-cardiac junction impairment in Duchenne muscular dystrophy.

doi: 10.1002/path.6362

Figure Lengend Snippet: Figure 2. MPs as main producers of CSPG4. (A) Graphic illustration of CSPG4-CAR-Ts cells against MPs expressing CSPG4. Created with Biorender.com. Diagrams quantifying the percentage of live cells of bone marrow-derived MPs of both WT and mdx M0 (nonactivated), M1 (pro-inflammatory) and M2 (anti-inflammatory), or CFs after exposure to CSPG4.CARTs compared to cells co-cultured with control CART (cCART). N = 3 biological replicates. (B) ELISA assay for CSPG4 in supernatants (SUP) isolated from WT and mdx MPs. The chart illustrates the optical density (OD) of CSPG4 in the two groups. Each dot represents the eluted fractions, N = 16. (C) FACSymphony of cardiac tissues. The first chart represents a scatter plot of t-SNE relative to concatenated total cells from 3- or 10-month-old WT and mdx hearts. The second chart illustrates the distribution of CD45neg cells (light pink cluster) and myeloid cells (dark pink cluster) in all groups. (D) Graph of FACS analysis relative to number of total MPs (F4/80) positive for CSPG4 in 3- or 10-month-old WT and mdx hearts. (E) Expression of CSPG4 in subpopulations of MPs: CD80pos (M1), CD206 pos (M2), Ly6Clow (resident), and Ly6Chigh (monocytes) in 3- or 10-month-old WT and mdx hearts. Error bars show SEM. *p < 0.05, **p < 0.01 calculated using Student’s t-test and one-way ANOVA.

Article Snippet: Western blot Proteins were extracted from tissues using lysis buffer (50 mM TRIS HCl pH 7.5), 0.6 M sucrose (Catalogue No. S0389, Sigma-Aldrich), 50% glycerol (Catalogue No. G5516, Sigma-Aldrich), 1% TRITON, 50 mM NaCl, 10 mM NaF, 2 mM sodium orthovanadate (Na3VO4, Catalogue No. 450243 Sigma-Aldrich), 1 mM PMSF (Catalogue No. 36978, Thermo Scientific,Waltham,MA, USA), 5 mM β-glycerophosphate (Catalogue No. G9422, Sigma-Aldrich), 1000X protease inhibitors (Catalogue No. 04693116001, Roche) for 30 min on ice, sonicated, and centrifuged at 12,000 g for 15 min at 4 C. Proteins were resolved using SDS-PAGE, transferred to nitrocellulose membranes (Catalogue No. GE10600079, Amersham Protran, Johns Creek, GA, USA), blocked with 5% nonfat dry milk powder (Catalogue No. 1.15363, Merck Millipore, Billerica, MA, USA) in PBS 1 for 45 min, and incubated at 4 C overnight with primary antibodies: tyrosine hydroxylase (1:1000, ab152), Synapsin 1 (1:1000, Cell Signaling Technology, #5297), CSPG4 (1:1000, Cell Signaling Technology, #52635), and Vinculin (1:500, V9131, Sigma-Aldrich), followed by incubation with secondary antibodies (antiRabbit NA934AV, 1:5000; anti-Mouse LNXa931/AE, 1:10000, GE Healthcare Life Sciences, Havelock, NE, USA) for 1 h at RT.

Techniques: Expressing, Derivative Assay, Cell Culture, Control, Enzyme-linked Immunosorbent Assay, Isolation

Journal: The Journal of pathology

Article Title: Macrophages producing chondroitin sulfate proteoglycan-4 induce neuro-cardiac junction impairment in Duchenne muscular dystrophy.

doi: 10.1002/path.6362

Figure Lengend Snippet: Figure 3. MPs release CSPG4 in media and inhibit axon growth of dorsal root ganglia. (A) Rendering of 3D NCJ generation. The picture was created with Biorender.com and illustrates the composition of the 3D NCJ model: WT dorsal root ganglia (DRG), WT cardiomyocytes (CMs), and WT CFs were encapsulated in PEG-FB hydrogel conditioned with the supernatants derived from MP cultures of WT, WT + CSPG4 blocking antibody (BAb), mdx, and mdx + CSPG4 (BAb). (B) Brightfield images of CFs, CMs, and DRG after 1 day of culture (upper panel) and 3D constructs of NCJ models after 7 days (lower panel). Scale bars, 100 μm. (C) Confocal images of 3D NCJ models: WT, WT + CSPG4 BAb, mdx, mdx + CSPG4 BAb. DRG are labeled with tubulin beta 3 (TUBB3, in yellow), CMs with cardiac troponin (cTNNT, in magenta), and CFs with fibroblast marker (ER- TR7, in white). (D) The graph indicates the area (%) occupied by neuron endings labeled by TUBB3 inside the 3D NCJ models. N = 3 biological replicates, N = 2 sections/constructs. Scale bars, 10 μm. **p < 0.01, ***p < 0.001 were determined using one-way ANOVA.

Article Snippet: Western blot Proteins were extracted from tissues using lysis buffer (50 mM TRIS HCl pH 7.5), 0.6 M sucrose (Catalogue No. S0389, Sigma-Aldrich), 50% glycerol (Catalogue No. G5516, Sigma-Aldrich), 1% TRITON, 50 mM NaCl, 10 mM NaF, 2 mM sodium orthovanadate (Na3VO4, Catalogue No. 450243 Sigma-Aldrich), 1 mM PMSF (Catalogue No. 36978, Thermo Scientific,Waltham,MA, USA), 5 mM β-glycerophosphate (Catalogue No. G9422, Sigma-Aldrich), 1000X protease inhibitors (Catalogue No. 04693116001, Roche) for 30 min on ice, sonicated, and centrifuged at 12,000 g for 15 min at 4 C. Proteins were resolved using SDS-PAGE, transferred to nitrocellulose membranes (Catalogue No. GE10600079, Amersham Protran, Johns Creek, GA, USA), blocked with 5% nonfat dry milk powder (Catalogue No. 1.15363, Merck Millipore, Billerica, MA, USA) in PBS 1 for 45 min, and incubated at 4 C overnight with primary antibodies: tyrosine hydroxylase (1:1000, ab152), Synapsin 1 (1:1000, Cell Signaling Technology, #5297), CSPG4 (1:1000, Cell Signaling Technology, #52635), and Vinculin (1:500, V9131, Sigma-Aldrich), followed by incubation with secondary antibodies (antiRabbit NA934AV, 1:5000; anti-Mouse LNXa931/AE, 1:10000, GE Healthcare Life Sciences, Havelock, NE, USA) for 1 h at RT.

Techniques: Derivative Assay, Blocking Assay, Construct, Labeling, Marker

Journal: The Journal of pathology

Article Title: Macrophages producing chondroitin sulfate proteoglycan-4 induce neuro-cardiac junction impairment in Duchenne muscular dystrophy.

doi: 10.1002/path.6362

Figure Lengend Snippet: Figure 4. Cardiac innervation in old mdx and WT mice. (A) Immunofluorescence staining for tyrosine hydroxylase (TH), Synapsin 1 (SYN1), total MPs (F4/80) and proteoglycan (CSPG4), fibronectin 1 (FN1) on cardiac sections from 10-month-old mice. The markers TH and SYN1 are labeled in yellow, while cardiac troponin (cTNNT) and F4/80 are in magenta, FN1 in white, and CSPG4 in yellow or white. DAPI nuclear counterstaining is in blue. Scale bars, 10 μm. (B) Charts indicate area expressed as percentage of TH, SYN1, and F4/80 (positive area/whole area). N = 3 sections per N = 3 biological replicates. (C and D) Western blot analysis for TH, SYN1, and CSPG4 quantified as optical density (OD) of protein bands normalized to vinculin (VCL). N = 3 biological replicates. (E) qPCR relative to markers for neurotransmitter production (Th), survival (Ngf, Ntrk1, and Ngfr) and axon guidance (Tubb3) in DRG. N = 3 biological replicates. Error bars show SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 were calculated using one-way ANOVA.

Article Snippet: Western blot Proteins were extracted from tissues using lysis buffer (50 mM TRIS HCl pH 7.5), 0.6 M sucrose (Catalogue No. S0389, Sigma-Aldrich), 50% glycerol (Catalogue No. G5516, Sigma-Aldrich), 1% TRITON, 50 mM NaCl, 10 mM NaF, 2 mM sodium orthovanadate (Na3VO4, Catalogue No. 450243 Sigma-Aldrich), 1 mM PMSF (Catalogue No. 36978, Thermo Scientific,Waltham,MA, USA), 5 mM β-glycerophosphate (Catalogue No. G9422, Sigma-Aldrich), 1000X protease inhibitors (Catalogue No. 04693116001, Roche) for 30 min on ice, sonicated, and centrifuged at 12,000 g for 15 min at 4 C. Proteins were resolved using SDS-PAGE, transferred to nitrocellulose membranes (Catalogue No. GE10600079, Amersham Protran, Johns Creek, GA, USA), blocked with 5% nonfat dry milk powder (Catalogue No. 1.15363, Merck Millipore, Billerica, MA, USA) in PBS 1 for 45 min, and incubated at 4 C overnight with primary antibodies: tyrosine hydroxylase (1:1000, ab152), Synapsin 1 (1:1000, Cell Signaling Technology, #5297), CSPG4 (1:1000, Cell Signaling Technology, #52635), and Vinculin (1:500, V9131, Sigma-Aldrich), followed by incubation with secondary antibodies (antiRabbit NA934AV, 1:5000; anti-Mouse LNXa931/AE, 1:10000, GE Healthcare Life Sciences, Havelock, NE, USA) for 1 h at RT.

Techniques: Staining, Labeling, Western Blot

Journal: The Journal of pathology

Article Title: Macrophages producing chondroitin sulfate proteoglycan-4 induce neuro-cardiac junction impairment in Duchenne muscular dystrophy.

doi: 10.1002/path.6362

Figure Lengend Snippet: Figure 5. Histological analysis and gene profile of cardiac tissue after treatment with givinostat in old mdx and WT mice. (A) Confocal images of immunostaining for gap junctions (CX43) and cardiomyocytes positive for cardiac troponin (cTNNT), N = 3 sections per N = 3 biological replicates. Scale bars, 50 μm. (B) Charts indicate area as percentage of CX43 (positive area/whole area). (C and D) Representative Masson’s trichrome images of WT, mdx saline, and mdx Giv hearts and fibrosis index quantification (fibrotic area/whole area) after 60 days of treatment. Scale bars, 100 μm. N = 3 biological replicates. (E) qPCR of fibrosis-related genes (Tgfb1, Col1a1, Col3a1, Twist1, Twist2, Fn1), cell surface proteoglycan (Cspg4); the enzyme catalyzes the transfer of sulfate groups on CSPG4 chains (Chst11) and inflammatory markers (Adgre1 and Mmp9). N = 3 biological replicates. Error bars show SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 were calculated using one-way ANOVA and Student’s t-test.

Article Snippet: Western blot Proteins were extracted from tissues using lysis buffer (50 mM TRIS HCl pH 7.5), 0.6 M sucrose (Catalogue No. S0389, Sigma-Aldrich), 50% glycerol (Catalogue No. G5516, Sigma-Aldrich), 1% TRITON, 50 mM NaCl, 10 mM NaF, 2 mM sodium orthovanadate (Na3VO4, Catalogue No. 450243 Sigma-Aldrich), 1 mM PMSF (Catalogue No. 36978, Thermo Scientific,Waltham,MA, USA), 5 mM β-glycerophosphate (Catalogue No. G9422, Sigma-Aldrich), 1000X protease inhibitors (Catalogue No. 04693116001, Roche) for 30 min on ice, sonicated, and centrifuged at 12,000 g for 15 min at 4 C. Proteins were resolved using SDS-PAGE, transferred to nitrocellulose membranes (Catalogue No. GE10600079, Amersham Protran, Johns Creek, GA, USA), blocked with 5% nonfat dry milk powder (Catalogue No. 1.15363, Merck Millipore, Billerica, MA, USA) in PBS 1 for 45 min, and incubated at 4 C overnight with primary antibodies: tyrosine hydroxylase (1:1000, ab152), Synapsin 1 (1:1000, Cell Signaling Technology, #5297), CSPG4 (1:1000, Cell Signaling Technology, #52635), and Vinculin (1:500, V9131, Sigma-Aldrich), followed by incubation with secondary antibodies (antiRabbit NA934AV, 1:5000; anti-Mouse LNXa931/AE, 1:10000, GE Healthcare Life Sciences, Havelock, NE, USA) for 1 h at RT.

Techniques: Immunostaining, Saline

Journal: Cell reports

Article Title: Pericyte dysfunction and impaired vasomotion are hallmarks of islets during the pathogenesis of type 1 diabetes

doi: 10.1016/j.celrep.2023.112913

Figure Lengend Snippet: Living pancreas slices were incubated with Fluo4 and NG2-Alexa647. (A) Confocal image of a pericyte in an islet from a NDdonor (nPOD6516) in basal glucose concentration (3 mM), upon high glucose (11 mM) alone, or in the presence of theophylline (20 μM; 11G + theo). Scale bar: 10 μm. (B) Heatmap showing changes in Fluo4 fluorescence (DF/F; %) in pericytes in islets from ND donors elicited by 11 mM glucose and then theo (20 μM; in 11G). Each line is a different pericyte. (C) Representative traces showing changes in Fluo4 fluorescence elicited by glucose and theo. (D) Net area under the curve (AUC) of fluorescence traces for individual pericytes. *p < 0.05 (paired t test; n = 113 pericytes from 5 ND donors). (E) Heatmaps showing changes in Fluo4 fluorescence in pericytes from ND (n = 9), Aab+ (n = 7), and T1D donors (n = 6). Donors are separated by white dashed lines. T1D donor data are presented based on T1D duration, while data from ND and Aab+ donors are presented according to . (F) Traces showing average changes in Fluo4 fluorescence elicited 11mM glucose for all islet pericytes from each group. Average (thicker lines) ± SEM values are shown. (G) Net AUC of fluorescence traces during stimulation with 11G (dots: data point for each donor, bars: mean ± SEM). For each donor, responses from around 3–36 pericytes were recorded, and an average value calculated. *p < 0.05 (one-sample t test, theoretical mean = 0).

Article Snippet: Living human pancreas slices were incubated with the cytosolic calcium indicator ([Ca 2+ ]i) Fluo4-AM (6 mM, Invitrogen, cat. nr. F14201) in 3 mM glucose solution prepared in HEPES buffer (125 mM NaCl, 5.9 mM KCl, 2.56 mM CaCl 2 , 1 mM MgCl2, 25 mM HEPES, 0.1% BSA [w/v], pH 7.4), supplemented with aprotinin (25 KIU), at room temperature and in the dark with either (a) DyLight 649 lectin from Lycopersicon Esculentum (3.3 mg/mL, VectorLabs, cat. nr. DL1178) for 1 hour; or with (b) a fluorescent-conjugated antibody against the pericyte marker neuron-glial antigen 2 (NG2-alexa647; 1:50, R&D Systems, cat. nr. Fab2585R) for 2 hours.

Techniques: Incubation, Concentration Assay, Fluorescence

Journal: Journal of Translational Medicine

Article Title: Identifying the functions of two biomarkers in human oligodendrocyte progenitor cell development

doi: 10.1186/s12967-021-02857-8

Figure Lengend Snippet: Identification of hOPC markers. a Three markers of hOPCs were identified using cell immunofluorescence staining: PDGFR-α, A2B5, and NG2. Scale bar is 200 μm. b Images of single cells detached in the presence of PDGFR-α+, A2B5+, and NG2+ cells, as observed using flow cytometry. Ch01: Bright field, Ch03: A2B5-PE, Ch07: PDGFR-α-BV421, Ch11: NG2-APC. Scale bar is 20 μm. c Gating strategy to define PDGFR-α/A2B5/NG2 cells. R4: PDGFR-α+ cells, R5: A2B5+ cells, R2: NG2+ cells. d scRNA-seq of hOPCs. Maps of t-SNE of 7885 cells from high-dimensional images of hOPCs coloured according to the cell type. hOPC human oligodendrocyte progenitor cells, PDGFR-α platelet-derived growth factor receptor alpha, scRNA-seq single-cell RNA sequencing

Article Snippet: The cells were surface-stained with PDGFR-α BV421 mouse anti-human (Cat. #562799, BD Biosciences, Franklin Lake, NJ, USA), NG2 APC mouse anti-human (Cat. #FAB2585A, R&D Systems, MN, USA), or A2B5 PE mouse anti-human (Cat. #130-093-581, Miltenyi Biotec, Bergisch-Gladbach, Germany) antibodies for 30 min at 4 °C.

Techniques: Immunofluorescence, Staining, Flow Cytometry, Derivative Assay, RNA Sequencing

Journal: Journal of Translational Medicine

Article Title: Identifying the functions of two biomarkers in human oligodendrocyte progenitor cell development

doi: 10.1186/s12967-021-02857-8

Figure Lengend Snippet: The expression of PDGFR-α in positive and negative cells. a Gating strategy to define PDGFR-α+ cells in A2B5+/− and NG2+/− populations. b Images of single cells detached in the presence of A + P +, A − P +, N + P +, and N − P + cells. c , d Cell counts of different cell populations. Bars represent the mean. Error bars show the standard error of the mean. A + P + , PDGFR-α+ cells in A2B5+ cells; A − P + , PDGFR-α+ cells in A2B5− cells; N + P + , PDGFR-α+ cells in NG2+ cells; N − P + , PDGFR-α+ cells in NG2− cells

Article Snippet: The cells were surface-stained with PDGFR-α BV421 mouse anti-human (Cat. #562799, BD Biosciences, Franklin Lake, NJ, USA), NG2 APC mouse anti-human (Cat. #FAB2585A, R&D Systems, MN, USA), or A2B5 PE mouse anti-human (Cat. #130-093-581, Miltenyi Biotec, Bergisch-Gladbach, Germany) antibodies for 30 min at 4 °C.

Techniques: Expressing

Journal: Journal of Translational Medicine

Article Title: Identifying the functions of two biomarkers in human oligodendrocyte progenitor cell development

doi: 10.1186/s12967-021-02857-8

Figure Lengend Snippet: Differential expression analysis. a Volcano plot illustrating differentially regulated gene expression between the four groups of cells. Red dots indicate upregulated genes, and blue dots indicate downregulated genes. Values are presented as the log2 of tag counts. b Heat map showing the relative gene expression in NG2+/− and A2B5+/− cells. Red and green indicate upregulated and downregulated genes, respectively. c , d Graph showing the FPKM value of markers in different cell populations. Bars represent the mean. Error bars show the standard error of the mean. FPKM fragments per kilobase of exon model per million mapped fragments

Article Snippet: The cells were surface-stained with PDGFR-α BV421 mouse anti-human (Cat. #562799, BD Biosciences, Franklin Lake, NJ, USA), NG2 APC mouse anti-human (Cat. #FAB2585A, R&D Systems, MN, USA), or A2B5 PE mouse anti-human (Cat. #130-093-581, Miltenyi Biotec, Bergisch-Gladbach, Germany) antibodies for 30 min at 4 °C.

Techniques: Quantitative Proteomics, Gene Expression

Journal: The Journal of Neuroscience

Article Title: Embryonic Pericytes Promote Microglial Homeostasis and Their Effects on Neural Progenitors in the Developing Cerebral Cortex

doi: 10.1523/jneurosci.1201-21.2021

Figure Lengend Snippet: Figure 2. Administration of APB5 induced pericytes to undergo apoptosis. A, A schematic showing the strategy of in vivo adminis- tration of anti-PDGFRb antibodies (APB5). B, Representative immunostaining for CD31 (green), NG2 (magenta), and PDGFRb (top) or PDGFRa (bottom; cyan) of the E14 cerebral wall. In top figures, white arrowhead indicates NG21PDGFRb 1 pericytes and yellow arrowhead indicates NG21PDGFRb – OPCs. In bottom figures, white arrowhead indicates NG21PDGFRa– pericytes and yellow arrowhead indicates NG21PDGFRa1 OPCs. C, Immunostaining for NG2 (green), CD31 (magenta), and DAPI (blue) in the E14 cerebral wall. D, A graph comparing the proportion of pericyte coverage, which was calculated by dividing the merged area of NG2 and CD31 by the total CD311 area in the pallium (not including the meninges), between the saline-treated, isotype control antibody-treated, and APB5-treated brains (two-sided Steel–Dwass test; the average value of four sections from each animal is plotted; N = 6 mice; p= 1.000 and p=0.011; left to right). Data are presented as the mean value 6 SD. E, Immunostaining for Cl-Casp3 (green), CD31 (magenta), and DAPI (blue) in the cerebral wall of the mice treated with antibodies or saline. The white broken line indicates the ap- ical surface. Scale bar: 50mm (B) or 100mm (C, E).

Article Snippet: Single cells were stained with rat FITC rat anti-mouse CD31 mAb (1:200, catalog #102405, BioLegend, RRID: AB_312900) or rat FITC IgG2a isotype control Ab (1:200, catalog #400505, BioLegend, RRID: AB_2736919), rat anti-NG2 mAb (1:200, catalog #MAB6689, R&D Systems, RRID: AB_10890940), which was labeled by PE using an Ab-10 Rapid R-Phycoerythrin Labeling kit (LK34, Dojindo), or rat PE IgG1 isotype control Ab (1:200, catalog #400408, BioLegend, RRID: AB_ 326514), and rat BV421 anti-PDGFRa mAb (1:200, catalog #135923, BioLegend, RRID: AB_2814036) or rat BV421 IgG2a isotype control antibody (1:200, catalog #407117, BioLegend, RRID: AB_2687343) for 1 h on ice.

Techniques: In Vivo, Immunostaining, Saline, Control

Journal: The Journal of Neuroscience

Article Title: Embryonic Pericytes Promote Microglial Homeostasis and Their Effects on Neural Progenitors in the Developing Cerebral Cortex

doi: 10.1523/jneurosci.1201-21.2021

Figure Lengend Snippet: Figure 6. An in vitro coculture showed that pericytes promoted microglial proliferation. A, FACS analysis of pallial cells collected from E14 mice to detect PDGFRb , NG2, and PDGFRa expres- sion. B, Pericyte sorting strategy by FACS. NG21PDGFRa– cells were collected from E14 cerebral wall cells. C, A schematic showing the bilayer culture of CD11b1 microglia with or without NG21PDGFRa– cells (pericytes) using the Transwell insert. D, Immunostaining for CX3CR1 and Ki67 in microglia cultured with or without NG21PDGFRa cells (pericytes). E, A bar graph com- paring the density of CX3CR11 cells (two-sided Mann–Whitney U test; the average value of six fields per independent culture is plotted; N = 6 independent cultures; p = 0.002). F, A bar graph comparing the proportion of Ki671 cells among CX3CR11 cells (two-sided Mann–Whitney U test; the average value of six fields per independent culture is plotted; N = 6 independent cultures; p = 0.002). G, Immunostaining for CX3CR1 and BrdU in microglia cultured with or without NG21PDGFRa cells (pericytes). Scale bar: 20mm. H, A bar graph comparing the proportion of BrdU1 cells among CX3CR11 cells (two-sided Mann–Whitney U test; the average value of six fields per independent culture is plotted; N = 6 independent cultures; p = 0.002). Data are pre- sented as the mean value 6 SD. Scale bar: 50 mm (D) or 20mm (G).

Article Snippet: Single cells were stained with rat FITC rat anti-mouse CD31 mAb (1:200, catalog #102405, BioLegend, RRID: AB_312900) or rat FITC IgG2a isotype control Ab (1:200, catalog #400505, BioLegend, RRID: AB_2736919), rat anti-NG2 mAb (1:200, catalog #MAB6689, R&D Systems, RRID: AB_10890940), which was labeled by PE using an Ab-10 Rapid R-Phycoerythrin Labeling kit (LK34, Dojindo), or rat PE IgG1 isotype control Ab (1:200, catalog #400408, BioLegend, RRID: AB_ 326514), and rat BV421 anti-PDGFRa mAb (1:200, catalog #135923, BioLegend, RRID: AB_2814036) or rat BV421 IgG2a isotype control antibody (1:200, catalog #407117, BioLegend, RRID: AB_2687343) for 1 h on ice.

Techniques: In Vitro, Immunostaining, Cell Culture, MANN-WHITNEY