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

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Nanoparticle Delivery of Proangiogenic Transcription Factors into the Neonatal Circulation Inhibits Alveolar Simplification Caused by Hyperoxia

doi: 10.1164/rccm.201906-1232OC

Figure Lengend Snippet: Polyethylenimine-(5) myristic acid/polyethylene glycol oleic acid/cholesterol (PEI600-MA5/PEG-OA/Cho) nanoparticles efficiently target endothelial cells in the neonatal lung. (A) Fluorescence-activated cell sorter (FACS) gating strategy to identify hematopoietic cells (Hema; CD45+CD31−), endothelial cells (Endo; CD31+CD45−CD326−), epithelial cells (Epi; CD326+CD45−CD31−), pericytes (Peric; NG2+PDGFRb+CD45−CD31−CD326−), and myofibroblasts (Myofibro; PDGFRa+CD45−CD31−CD326−). DyLight 650–labeled nanoparticles were delivered at Postnatal Day 2 (P2). FACS analysis of enzymatically digested lung tissue was performed at P5. (B and C) Dot plots show the presence of nanoparticles in different populations of pulmonary cells. Noninjected mice were used as control animals to identify cells containing nanoparticles. (D) Percentage of nanoparticle-targeted cells is shown among pericytes; myofibroblasts; and epithelial, endothelial, and hematopoietic cells (n = 4 mice per group). Error bars are mean ± SE. NG2 = melanoma-associated chondroitin sulfate proteoglycan 4; PDGFRα = platelet-derived growth factor receptor-α; PDGFRβ = platelet-derived growth factor receptor-β.

Article Snippet: Cells not expressing any of these markers (CD45 – CD31 – CD326 – ) were then evaluated for pericytes (PDGFRb + NG2 + CD45 – CD31 – CD326 – ) using PDGFRb (platelet-derived growth factor receptor-β) (CD140b) Ab (clone APB5; BioLegend) and NG2 (melanoma-associated chondroitin sulfate proteoglycan 4) Ab (clone 9.2.27; BD Biosciences).

Techniques: Fluorescence, Labeling, Derivative Assay

Journal: bioRxiv

Article Title: Reduced myofibroblast transdifferentiation and fibrotic scarring in ischemic stroke after imatinib treatment

doi: 10.1101/2021.10.28.466225

Figure Lengend Snippet: (a, b) Immunofluorescent staining for the astrocyte marker GFAP (red) was performed on brain sections from imatinib and vehicle-treated mice 3 hours post MCAO. (a) Stitched tiles of epifluorescent images (taken with a 10x objective). (b) Confocal images of the boxed regions in (a) show that imatinib reduced astrocyte activation in the ischemic area while preserving vascular associated GFAP + staining (arrows). (c) Quantification of GFAP signal in the ischemic area based on six confocal images per brain. (d, e) Immunofluorescent staining for PDGFR α (green) show that the cell body area of PDGFR α + NG2-glia cells (e, red) are increased in the ischemic border of the ipsilateral (ipsi) hemisphere 3 hours post MCAO in vehicle controls (two headed arrows) compared to the contralateral hemisphere and imatinib-treated mice (arrows). (f) Quantification of PDGFR α + cell body area in the ischemic border based on ten confocal images per brain. (g) Immunofluorescence analysis of microglia activation was performed by staining for CD11b (green) on brain sections from imatinib-treated mice and vehicle controls at different times after MCAO. Microglia (arrows); activated microglia/macrophages (two headed arrows). (h) Quantification of activated microglia/macrophages was determined in the whole ischemic area in four sections per animal. ND; not detected. (i) qPCR analysis of RNA isolated from vascular fragments harvested 3 hours, 24 hours and 7 days post MCAO. Il1a, Tnfa and Ccl2 are shown. Symbols represent individual data points and bars the group mean ± S.E.M. Cell nuclei were visualized by DAPI and endothelial cells by CD31. Statistical significance *p < 0.05; **p < 0.01; *** p < 0.001; in i) # refer to statistics between imatinib and vehicle and * to statistics between sham and MCAO (c, f, Student’s unpaired t-test; h, i, one-way ANOVA with Fisher’s LSD test). Representative maximum intensity projections of 11 µm (d); 8 µm (e); 12 µm (g) z-stacks are shown. Scale bars, 1000 µm (a); 50 µm (b); 25 µm (d); 10 µm (e, g).

Article Snippet: The specific primary antibodies used were: goat anti-mouse PDGFR α (AF1062, R&D systems), rabbit anti-mouse GFAP (Z0334, DAKO), rat anti-GFAP (clone 2.2B10, 13-0300, Invitrogen), rabbit anti-rat NG2 (AB5320, Merck/Millipore), mouse anti- α Smooth Muscle Actin-Cy3 (ASMA, C6198, Sigma-Aldrich), goat anti-mouse CD31 (AF3628, R&D systems), rat anti-mouse CD31 (553370, BD Pharmingen), rat anti-mouse CD11b (550282, BD Pharmingen), rabbit anti-mouse CD3 (C7930, Sigma-Aldrich), rabbit anti-mouse MPO (ab208670, Abcam), rat anti-mouse B220 (CD45R, MAB1217, R&D systems), rabbit anti-fibronectin (F3648, Sigma-Aldrich) and rabbit anti-aquaporin 4 (AB2218, Merck/Millipore).

Techniques: Staining, Marker, Activation Assay, Preserving, Immunofluorescence, Isolation

Journal: bioRxiv

Article Title: Reduced myofibroblast transdifferentiation and fibrotic scarring in ischemic stroke after imatinib treatment

doi: 10.1101/2021.10.28.466225

Figure Lengend Snippet: Immunofluorescent co-stainings of PDGFR α with GFAP or NG2. (a – b) Stitched tiles of epifluorescent images (taken with a 10x objective). (c) Imatinib treatment reduced the formation of the PDGFR α + scar 7 days post MCAO (arrows) compared to vehicle controls (two headed arrows), without affecting the thickness of the GFAP + (a) or NG2 + (b) scar (demarcated with dashed lines). Quantification of PDGFR α + scar thickness was determined by 20 individual measures along the scar. (d) Confocal images of the staining in (b) showing that MCAO induced ectopic expression of PDGFR α in NG2 - non-perivascular cells (two headed arrows) as opposed to normal expression in NG2 + glia cells (arrows). Symbols represent individual data points and bars the group mean ± S.E.M. Statistical significance *p < 0.05 relative to control (Student’s unpaired t-test). Representative maximum intensity projections of 11 µm z-stacks are shown in (d). Scale bars, 1000 µm (a, b); 25 µm (d).

Article Snippet: The specific primary antibodies used were: goat anti-mouse PDGFR α (AF1062, R&D systems), rabbit anti-mouse GFAP (Z0334, DAKO), rat anti-GFAP (clone 2.2B10, 13-0300, Invitrogen), rabbit anti-rat NG2 (AB5320, Merck/Millipore), mouse anti- α Smooth Muscle Actin-Cy3 (ASMA, C6198, Sigma-Aldrich), goat anti-mouse CD31 (AF3628, R&D systems), rat anti-mouse CD31 (553370, BD Pharmingen), rat anti-mouse CD11b (550282, BD Pharmingen), rabbit anti-mouse CD3 (C7930, Sigma-Aldrich), rabbit anti-mouse MPO (ab208670, Abcam), rat anti-mouse B220 (CD45R, MAB1217, R&D systems), rabbit anti-fibronectin (F3648, Sigma-Aldrich) and rabbit anti-aquaporin 4 (AB2218, Merck/Millipore).

Techniques: Staining, Expressing

Journal: bioRxiv

Article Title: Reduced myofibroblast transdifferentiation and fibrotic scarring in ischemic stroke after imatinib treatment

doi: 10.1101/2021.10.28.466225

Figure Lengend Snippet: To the left, the NVU during normoxia. The endothelium maintains a tight barrier with the support of the basement membrane (BM), vascular mural cells (including vascular smooth muscle cells, vSMC) and perivascular astrocytes and fibroblast like cells (FB). During normoxia non-vascular astrocytes, NG2 glia and microglia are ramified. To the right, the NVU and cellular responses during hypoxia after MCAO without (depicted above vessel) or with (depicted below vessel) imatinib treatment. In the acute phase (hours) after onset of hypoxia, MCAO induces reorganization in the NVU, associated with increased vascular permeability, as well as an early reactive gliosis response, including activation of astrocytes, NG2 glia and microgliosis/macrophage recruitment. This is dampened by imatinib treatment and is supported by BBB transcriptome analyses showing that imatinib treatment downregulate expression of proinflammatory and profibrotic transcripts including Ccl2, Ccl5, Cdh11, Cntfr and Sirpb1 . In the tissue remodeling subacute/chronic phase after MCAO (days), imatinib reduces transdifferentiation/expansion of myofibroblasts and PDGFR α fibrotic scar formation. Taken together this results in a progressive improvement in functional outcome.

Article Snippet: The specific primary antibodies used were: goat anti-mouse PDGFR α (AF1062, R&D systems), rabbit anti-mouse GFAP (Z0334, DAKO), rat anti-GFAP (clone 2.2B10, 13-0300, Invitrogen), rabbit anti-rat NG2 (AB5320, Merck/Millipore), mouse anti- α Smooth Muscle Actin-Cy3 (ASMA, C6198, Sigma-Aldrich), goat anti-mouse CD31 (AF3628, R&D systems), rat anti-mouse CD31 (553370, BD Pharmingen), rat anti-mouse CD11b (550282, BD Pharmingen), rabbit anti-mouse CD3 (C7930, Sigma-Aldrich), rabbit anti-mouse MPO (ab208670, Abcam), rat anti-mouse B220 (CD45R, MAB1217, R&D systems), rabbit anti-fibronectin (F3648, Sigma-Aldrich) and rabbit anti-aquaporin 4 (AB2218, Merck/Millipore).

Techniques: Permeability, Activation Assay, Expressing, Functional Assay

Journal: eLife

Article Title: O-GlcNAc on NOTCH1 EGF repeats regulates ligand-induced Notch signaling and vascular development in mammals

doi: 10.7554/elife.24419

Figure Lengend Snippet: Figure 7. Reduced vessel integrity in the Eogt/ retina. (A) Immunostaining with fibrinogen (green) and a-SMA (magenta) antibodies in P15 wild-type, Eogt/, Tek-Cre, Tek-Cre:EogtF/F, Notch1+/, and Rbpj+/ retinas. Arrows indicate fibrinogen staining outside vessels stained by IB4 (white). Three- dimensional images were constructed from confocal images by maximum intensity projection. (B) Higher magnification three-dimensional images of Eogt/ retina constructed from confocal images using the Alpha-blend method. Below, single channel images showing fibrinogen (green) and IB4 (white) staining. (C) Sulfo-NHS-LC-biotin was perfused into P15 wild-type and Eogt/ mice and extravasation determined immediately after perfusion by staining with CF488A-conjugated streptavidin (green) and Dylight594-conjugated IB4 (white). Three-dimensional reconstructions were created by maximum intensity projection. Enlarged images of boxed area are shown (right). (D) Sulfo-NHS-LC-biotin was perfused into P15 wild-type, Eogt/, Notch1+/, Eogt/Notch1+/ mice as in (C). Quantification of the number of extravasation sites in 210 210 mm squares (n = 6 per retina per mouse) is shown. Note that sulfo-NHS-LC-biotin extravasation in Eogt/ retina is augmented in compound mutant mice. Data represent mean ± standard error; p values determined by Welch’s t test. ***p0.001. (E) Whole-mount images of wild-type or Eogt/ P15 retinas stained with IB4 (cyan) and anti-a SMA (magenta) antibody. (F) Whole-mount staining of wild-type and Eogt/ P15 retinas using IB4 (white) together with anti-fibrinogen (green) and anti-NG2 (magenta) antibodies. DOI: 10.7554/eLife.24419.018 The following source data and figure supplement are available for figure 7:

Article Snippet: Antibodies used in microscopy, flow cytometry and Western blot experiments: biotinylated isolectin B4 (IB4; Vector [B-1105]), Cy3-conjugated anti–a-smooth muscle actin (aSMA) antibody (clone 1A4; Sigma [C6198] or fluorescein isothiocyanate(FITC)-conjugated anti-aSMA antibody (clone 1A4; Sigma [F3777]), rabbit anti-human EOGT antibody (Sigma [HPA019460]), mouse anti-O-GlcNAc antibody (CTD110.6; Thermo Scientific [24565] or Sigma [07764]) (Comer et al., 2001), hamster antimouse NOTCH1 ECD antibody (8G10, Santa Cruz [sc-32756]), sheep anti-hamster NOTCH1 ECD antibody (R and D Systems, AF5267), rabbit anti-human NOTCH1 ECD antibody (H-131, Santa Cruz [sc-9170]), rabbit anti-human NOTCH1 ICD antibody (D6F11, Cell signaling), rabbit anti-mouse activated NOTCH1 (Val1744, Cell Signaling Technology [4147]) (Huppert et al., 2000), sheep anti-BiP antibody (BD Biosciences [51–9001980]), rabbit anti-NG2 chondroitin sulfate antibody (Millipore [AB5320]), rat anti-NG2 chondroitin sulfate antibody (R and D Systems [MAB6689]), mouse anti-Myc antibody (4A6, Upstate [05-724]), hamster anti-mouse JAG1 antibody (HMJ1-29; Biolegend [130902]), goat anti-mouse DLL4 antibody (R and D Systems [AF1389]), rabbit anti-fibrinogen antibody (Dako [A0080]), FITC-conjugated anti-hamster IgG antibody (Cappel [55400]), CF488A-conjugated anti-goat IgG antibody (Sigma [SAB4600032]), Dylight488–conjugated anti-rabbit IgG (Vector Labs [DI-1488]), CF488A-conjugated streptavidin (Biotium [29034]), CF640R-conjugated anti-rat IgG (Sigma [SAB4600156]), Rhodamine Red-X-conjugated donkey anti-sheep IgG (Jackson ImmunoResearch), Dylight649-conjugated streptavidin (Vector Labs [SA5649]), horseradish peroxidase (HRP)conjugated goat anti-mouse IgM (Thermo Scientific [31444]), HRP-conjugated goat anti-rabbit IgG (Invitrogen [65–6120]), HRP-conjugated goat anti-Armenian hamster IgG (Santa Cruz [sc-2443]), HRPconjugated horse anti-mouse IgG antibody (Cell signaling [7076S]), alkaline phosphatase (AP)-conjugated anti-FITC antibody (Roche [11-426-338-910]), FITC-conjugated IB4 (Vector Labs [FL-1201]), and Dylight594-conjugated IB4 (Vector Labs [DL-1178]).

Techniques: Immunostaining, Staining, Construct, Mutagenesis