Journal: Nucleic Acids Research

Article Title: Heterochromatome wide analyses reveal MBD2 as a phase separation scaffold for heterochromatin compartmentalization and composition

doi: 10.1093/nar/gkaf1380

Figure Lengend Snippet: MBD2 is a candidate phase separation scaffold protein in mouse pericentric heterochromatin. (A) Number of predicted phase separation scaffold proteins with GO terms for multiple subnuclear membraneless organelles. (B) Venn diagram showing the overlap of three independent proteome-wide data to identify the most likely phase separation (PS) scaffold proteins in pericentric heterochromatin (PCH). “PCH Prediction” represented all predicted phase separation scaffold proteins with GO terms for pericentric heterochromatin compartment, as shown in panel (A). “HC_P” represents all proteins identified in the pellet fraction after heterochromatin phase separation and DIA_MS as shown in Fig. . “PCH proteome” represents the pericentric heterochromatin proteomic data from Schmidt et al. . (C) Table list of published (Green) and newly identified (black) phase separation scaffold proteins (PS scaffolds) in pericentric heterochromatin as shown in panel (B). PdPS, partner-dependent phase separation; SaPS, self-assembling phase separation. (D) Scheme summarizing the structures, length (aa), disorder scores, and isoelectric points (pI) of MBD-containing proteins. MBD: methyl-CpG binding domain; TRD: transcriptional repression domain. G/R: glycine/arginine; CC: coiled coil domain. The disorder scores were predicted using IUPred2A ( https://iupred2a.elte.hu/ ). The isoelectric points (pI) were calculated using the peptide property calculator ( https://pepcalc.com/ ). ( E, F ) Representative DIC images showing the phase separation properties of MBD-containing proteins. Following in vitro phase separation , the mixtures were transferred to chambers made of double-sided tapes and sealed with coverslips 45 min after incubation at room temperature. The droplets were observed using a Nikon Eclipse TiE2 microscope equipped with DIC microscopy. Images were taken using a Nikon Eclipse TiE2 microscope equipped with DIC. n = three replicates. Scale bar = 10 μm.

Article Snippet: 2D images were taken using Leica TCS SP5 II confocal microscope with a HCX PL APO 100×/1.44 oil Corr CS objective or Nikon Eclipse TiE2 microscope equipped with a Plan Apo λ 40× air objective ( ).

Techniques: Binding Assay, In Vitro, Incubation, Microscopy

Journal: Nucleic Acids Research

Article Title: Heterochromatome wide analyses reveal MBD2 as a phase separation scaffold for heterochromatin compartmentalization and composition

doi: 10.1093/nar/gkaf1380

Figure Lengend Snippet: MBD2a/b influences chromatin compaction. (A) Schematic representation of MBD2 isoforms and constructs used for ectopic expression. (B) Representative images of ectopic MBD2a and total MBD2 distribution within nuclei. Total MBD2 levels were detected by immunofluorescence staining using antibody against the MBD domain of MBD2 and Cy5-conjugated secondary antibody, followed by fluorescence imaging using a Nikon Eclipse TiE2 microscope equipped with a Plan Apo λ 40× air objective. ( C, D ) Heat map showing the influence of MBD2 isoforms on their subnuclear localization (C) and pericentric heterochromatin compartment sizes (D) with and without CC domain. Nuclei were subclassified into four categories based on their total MBD2 levels (I: endogenous, 2–3 µM as calculated in ). Assuming a linear relationship of Cy5 fluorescence and total MBD2 levels, we estimated the protein levels (µM) of each nucleus and subgrouped them based on their MBD2 levels: II 20 µM (10–30 µM); III 40 µM (30–50 µM); and IV > 50 µM. For the heat map, mean values were plotted. Cell numbers in each condition were shown in panel (C). Significances were determined by pairwise t -test with Benjamini–Hochberg (BH) correction false discovery rate (FDR) correction. ns, no significance, P >.05; * P ≤.05; ** P ≤.01; *** P ≤.001. Raw data can be found in . (E) Bar plot showing the effect of the different MBD2 isoforms on the compaction of chromatin. The cell nucleus was classified into seven different chromatin compaction classes based on the DAPI intensities from DNA-free interchromatin region (class 1) to highly active and less compacted euchromatin (classes 2–4) and to highly compacted heterochromatin (classes 5–7). 3D images were taken using Leica TCS SP5 II confocal microscope with a HCX PL APO 100×/1.44 oil Corr CS objective. 3D images with similar MBD2 protein levels were taken and used for quantitative analysis. Data are represented as mean ± SD. n (control) = 33. n (MBD2a) = 32. n (MBD2b) = 34. n (MBD2c) = 32. Significances were calculated by an unpaired t -test. ns, no significance, P >.05; * P ≤.05; ** P ≤.01; *** P ≤.001. Raw data can be found in .

Article Snippet: 2D images were taken using Leica TCS SP5 II confocal microscope with a HCX PL APO 100×/1.44 oil Corr CS objective or Nikon Eclipse TiE2 microscope equipped with a Plan Apo λ 40× air objective ( ).

Techniques: Construct, Expressing, Immunofluorescence, Staining, Fluorescence, Imaging, Microscopy, Control

Journal: Nucleic Acids Research

Article Title: Heterochromatome wide analyses reveal MBD2 as a phase separation scaffold for heterochromatin compartmentalization and composition

doi: 10.1093/nar/gkaf1380

Figure Lengend Snippet: MBD2a/b decreases the histone acetylation levels in pericentric heterochromatin regions in both C2C12 ( A, B ) and ES ( C–F ) cells. ( A, B ) Representative images (top) and quantitative analysis of histone acetylation [H3K9ac (A) , H3K27ac (B) ] abundance in whole nuclei and in pericentric heterochromatin (PCH) (bottom) in C2C12 cells 48 h after transfection of plasmid coding for GFP-MBD2 isoforms. Cells with similar expression levels of MBD2 isoforms were chosen. H3K9ac and H3K27ac were visualized by immunofluorescence staining after transfection. DNA was counterstained with DAPI. Images were taken using a Nikon Eclipse TiE2 microscope equipped with a Plan Apo λ 40× air objective. Data are represented as scatter plots with mean ± SD. The red dashed line indicates the mean fluorescence intensities in cells with GFP expression. For panel (A), n (MBD2a) = 172; n (MBD2b) = 145; n (MBD2c) = 205; n (GFP) = 919. For panel (B), n (MBD2a) = 1459; n (MBD2b) = 831; n (MBD2c) = 3016; n (GFP) = 5779. Significances were calculated by an unpaired t -test. ns P >.05; *** P ≤.001. Raw data can be found in . ( C, D ) Representative images (top) and quantitative analysis of histone acetylation [H3K9ac (C) , H3K27ac (D) ] abundance in whole nuclei and in pericentric heterochromatin (PCH) (bottom) in ES cells 48 h after transfection of plasmid coding for GFP-MBD2 isoforms. Cells were classified into four classes based on GFP intensities. Each class contains cells with similar GFP intensities. H3K9ac and H3K27ac were visualized by immunofluorescence staining after transfection. DNA was counterstained with DAPI. Images were taken using a Nikon Eclipse TiE2 microscope equipped with a Plan Apo λ 40× air objective. Data are represented as heat maps showing the mean values. Cell numbers in each condition were shown. Raw data can be found in . ( E-F ) Representative images (top) and quantitative analysis of histone acetylation [H3K9ac (E) , H3K27ac (F) ] abundance in whole nuclei and in pericentric heterochromatin (PCH) (bottom) in wild-type (WT) and MBD2 knockout (MBD2-KO) ES cells. H3K9ac and H3K27ac were visualized by immunofluorescence staining. DNA was counterstained with DAPI. Images were taken using a Nikon Eclipse TiE2 microscope equipped with a Plan Apo λ 40× air objective. Data are represented by violin plots embedded with box plots. Violin plots displayed the probability density of the data at different values, mirrored around the center line. The box plots indicated the median (central line), interquartile range (IQR) (box), and whiskers representing 1.5 × IQR. For panel (E), n (WT) = 318; n (MBD2-KO) = 255. For panel (F), n (WT) = 148; n (MBD2-KO) = 186. Raw data can be found in .

Article Snippet: 2D images were taken using Leica TCS SP5 II confocal microscope with a HCX PL APO 100×/1.44 oil Corr CS objective or Nikon Eclipse TiE2 microscope equipped with a Plan Apo λ 40× air objective ( ).

Techniques: Transfection, Plasmid Preparation, Expressing, Immunofluorescence, Staining, Microscopy, Fluorescence, Knock-Out

Journal: Stem cells (Dayton, Ohio)

Article Title: Human bone marrow-derived mesenchymal stem cells suppress human glioma growth through inhibition of angiogenesis.

doi: 10.1002/stem.1247

Figure Lengend Snippet: Figure 4. Conditioned medium from DGli36/MSC coculture prevented recruitment of EPC and endothelial tube formation. (A): EPCs were immunostained with antibodies against CD133, CD34, VE-cadherin, VEGFR-2, Tie2, and CD45. Mouse IgG1 and rabbit serum were used as neg- ative control for the first five antibodies and CD45, respectively. Scale bar ¼ 100 lm. (B): EPC and MSC were assessed for their ability to uptake DiI-Ac-LDL (red fluorescence). Cells were counterstained with DAPI (blue fluorescence). Representative images were presented. Scale bar ¼ 50 lm. (C): EPC recruitment assay was performed using DGli36-CM, MSC-CM, and DGli36/MSC-CM. The number of recruited cells to the bottom chamber were counterstained with propidium iodide and counted. Data shown are averages of triplicates 6 SEM. *, p < .05. (D): Angiogenesis assay was performed to determine whether DGli36-CM, MSC-CM, NHA-CM, DGli36/NHA-CM, or DGli36/MSC-CM could induce tube formation of HUVEC. 50 ng/ml of recombinant human VEGF was used as positive control. Tubes formed were visualized and captured using the Zeiss 200M wide-field microscope at 50 original magnification. Scale bar ¼ 200 lm. (E): Tube formation index was scored using ImageJ software (National Institutes of Health, Bethesda, MD). Data were obtained from four replicates of two independent experiments and pre- sented as ratio of coculture to monoculture. Abbreviations: CM, conditioned medium; EPC, endothelial progenitor cells; LDL, low density lipo- protein; NHA, normal human astrocytes; MSC, mesenchymal stem cell; VE, vascular endothelial; VEGFR, vascular endothelial growth factor receptor; DAPI, 40,6-diamidino-2-phenylindole; HUVEC, Human umbilical vein endothelial cells.

Article Snippet: The following antibodies were used for the various staining: CD31 (1 lg/ml; BD Biosciences), cathepsin B (5 lg/ml; R&D Systems, Minneapolis, MN, http://www.rndsystems.com), single-stranded DNA (ssDNA; 1 lg/ml; clone F7-26, Millipore, Temecula, CA, http:// www.millipore.com), a-smooth muscle actin (SMA; 1:100 dilution; Sigma Aldrich, St. Louis, MO, http://www.sigmaaldrich. com), CD34 (1 lg/ml), CD133 (1:100 dilution), Tie2 (1 lg/ml), VE-cadherin (1 lg/ml) and VEGFR-2 (1 lg/ml) (Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com), and CD45 (1:100 dilution; BD Biosciences).

Techniques: Control, Angiogenesis Assay, Recombinant, Positive Control, Microscopy, Software

Journal: Neural Regeneration Research

Article Title: Structural and functional damage to the hippocampal neurovascular unit in diabetes-related depression

doi: 10.4103/1673-5374.244794

Figure Lengend Snippet: Diagrams of the in vitro NVU system and the double cell co-culture systems. Astrocytes were seeded in the outer side of the matching Transwell inserts after neurons were seeded into a 24-well culture plate and cultured for 5–7 days. After 2 days of co-culture, endothelial cells were seeded in the inner side of the matching well inserts. The NVU triple cell system and the double cell co-culture systems were successfully established after 3 days of culture. NE: Neuron; AS: astrocyte; BM: brain microvascular endothelial cell; NO: no cell; NVU: neurovascular unit.

Article Snippet: Astrocytes and brain microvascular endothelial cells were labeled with rabbit anti-glial fibrillary acidic protein (GFAP) (1:100; Boster) and rabbit anti-PECAM-1/CD31 (1:100; Boster) antibodies.

Techniques: In Vitro, Co-Culture Assay, Cell Culture

Journal: Neural Regeneration Research

Article Title: Structural and functional damage to the hippocampal neurovascular unit in diabetes-related depression

doi: 10.4103/1673-5374.244794

Figure Lengend Snippet: Morphological and immunocytochemical characteristics of the hippocampal NVU. (A) Hippocampal neurons, astrocytes and microvascular endothelial cells under an inverted light microscope. (B) Neurons are positive for NSE (arrows), astrocytes express GFAP (arrows), and brain microvascular endothelial cells are positive for CD31 (arrows). Scale bars: 100 μm. NVU: Neurovascular unit; NSE: neuron-specific enolase; GFAP: glial fibrillary acidic protein.

Article Snippet: Astrocytes and brain microvascular endothelial cells were labeled with rabbit anti-glial fibrillary acidic protein (GFAP) (1:100; Boster) and rabbit anti-PECAM-1/CD31 (1:100; Boster) antibodies.

Techniques: Light Microscopy

Journal: Neural Regeneration Research

Article Title: Structural and functional damage to the hippocampal neurovascular unit in diabetes-related depression

doi: 10.4103/1673-5374.244794

Figure Lengend Snippet: Expression of occludin protein in the hippocampal NVU triple cell co-culture system and in the AS + BM co-culture system, with or without glucose (150 mM) and corticosterone (200 μM), assessed by western blot assay. Results are shown as the mean ± SD ( n = 3; one-way analysis of variance followed by Dunnett’s post hoc test). * P < 0.05, ** P < 0.01, vs . NVU group; # P < 0.05, vs . NVU model group (NVU + glucose + corticosterone). Western blot assay was performed in triplicate. GAPDH was used as loading control. The numbers on the right are the molecular weights of the proteins (kDa). AS: Astrocyte; BM: brain microvascular endothelial cell; NVU: neurovascular unit; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Astrocytes and brain microvascular endothelial cells were labeled with rabbit anti-glial fibrillary acidic protein (GFAP) (1:100; Boster) and rabbit anti-PECAM-1/CD31 (1:100; Boster) antibodies.

Techniques: Expressing, Co-Culture Assay, Western Blot, Control

Journal: Neural Regeneration Research

Article Title: Structural and functional damage to the hippocampal neurovascular unit in diabetes-related depression

doi: 10.4103/1673-5374.244794

Figure Lengend Snippet: Transendothelial electrical resistance (A) and leakage liquid height (B) in the hippocampal NVU triple cell co-culture system and in the AS + BM co-culture system, with or without glucose (150 mM) and corticosterone (200 μM), as detected by Millipore resistor and vernier caliper. Quantitative results are shown as the mean ± SD ( n = 3; one-way analysis of variance followed by Dunnett’s post hoc test). * P < 0.05, ** P < 0.01, vs . NVU group; ## P < 0.01, vs . NVU model group (NVU + glucose + corticosterone); $ P < 0.05, $$ P < 0.01, vs . AS + BM group. Experiments were performed in triplicate. NE: Neuron; AS: astrocyte; BM: brain microvascular endothelial cell; NVU: neurovascular unit; TEER: transendothelial electrical resistance.

Article Snippet: Astrocytes and brain microvascular endothelial cells were labeled with rabbit anti-glial fibrillary acidic protein (GFAP) (1:100; Boster) and rabbit anti-PECAM-1/CD31 (1:100; Boster) antibodies.

Techniques: Co-Culture Assay

Journal: Neural Regeneration Research

Article Title: Structural and functional damage to the hippocampal neurovascular unit in diabetes-related depression

doi: 10.4103/1673-5374.244794

Figure Lengend Snippet: Expression of CX43 and GFAP proteins in the hippocampal NVU triple cell co-culture system and in the AS + BM co-culture system, with or without glucose (150 mM) and corticosterone (200 μM), as detected by western blot assay. Results are shown as the mean ± SD ( n = 3; one-way analysis of variance followed by Dunnett’s post hoc test). * P < 0.05, ** P < 0.01, vs . NVU group; # P < 0.05, vs . NVU model group (NVU + glucose + corticosterone). Western blot assay was performed in triplicate. GAPDH was used as loading control. Numbers on the right indicate the molecular weights of the proteins (kDa). NE: Neuron; AS: astrocyte; BM: brain microvascular endothelial cell; NVU: neurovascular unit; CX43: connexin 43; GFAP: glial fibrillary acidic protein.

Article Snippet: Astrocytes and brain microvascular endothelial cells were labeled with rabbit anti-glial fibrillary acidic protein (GFAP) (1:100; Boster) and rabbit anti-PECAM-1/CD31 (1:100; Boster) antibodies.

Techniques: Expressing, Co-Culture Assay, Western Blot, Control

Journal: Neural Regeneration Research

Article Title: Structural and functional damage to the hippocampal neurovascular unit in diabetes-related depression

doi: 10.4103/1673-5374.244794

Figure Lengend Snippet: Intracellular and supernatant levels of 5-HT in the hippocampal NVU triple cell co-culture system and in the AS + BM co-culture system, with or without glucose (150 mM) and corticosterone (200 μM), as measured by enzyme-linked immunosorbent assay. Results are shown as the mean ± SD ( n = 3; one-way analysis of variance followed by Dunnett’s post hoc test). ** P < 0.01, vs . NVU group; ## P < 0.01, vs . NVU model group (NVU + glucose + corticosterone); $ P < 0.05, vs . AS + BM group. Experiments were performed in triplicate. NE: Neuron; AS: astrocyte; BM: brain microvascular endothelial cell; NVU: neurovascular unit; F&M: fluoxetine and metformin; 5-HT: 5-hydroxytryptamine.

Article Snippet: Astrocytes and brain microvascular endothelial cells were labeled with rabbit anti-glial fibrillary acidic protein (GFAP) (1:100; Boster) and rabbit anti-PECAM-1/CD31 (1:100; Boster) antibodies.

Techniques: Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: Neural Regeneration Research

Article Title: Structural and functional damage to the hippocampal neurovascular unit in diabetes-related depression

doi: 10.4103/1673-5374.244794

Figure Lengend Snippet: Apoptosis in the hippocampal NVU triple cell co-culture system and in the AS + BM co-culture system, with or without glucose (150 mM) and corticosterone (200 μM), as detected by flow cytometry. Results are shown as the mean ± SD ( n = 3; one-way analysis of variance followed by Dunnett’s post hoc test). ** P < 0.01, vs . NVU group; # P < 0.05, vs . NVU model group (NVU + G & P, NVU + glucose + corticosterone). All apoptotic cells were in the Q2 and Q4 quadrants. Early apoptotic cells were in Q4, while late apoptotic cells were in Q2. Experiments were performed in triplicate. NE: Neuron; AS: astrocyte; NVU: neurovascular unit; G&P: glucose and corticosterone; F&M: fluoxetine and metformin.

Article Snippet: Astrocytes and brain microvascular endothelial cells were labeled with rabbit anti-glial fibrillary acidic protein (GFAP) (1:100; Boster) and rabbit anti-PECAM-1/CD31 (1:100; Boster) antibodies.

Techniques: Co-Culture Assay, Flow Cytometry

Journal: Neural Regeneration Research

Article Title: Structural and functional damage to the hippocampal neurovascular unit in diabetes-related depression

doi: 10.4103/1673-5374.244794

Figure Lengend Snippet: Apoptosis in the hippocampal NVU triple cell co-culture system and in the AS + BM co-culture system, with or without glucose (150 mM) and corticosterone (200 μM), as detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling staining. Arrows point to apoptotic cells. NE: Neuron; AS: astrocyte; NVU: neurovascular unit; G & P: glucose and corticosterone; F & M: fluoxetine and metformin. Scale bars: 100 μm.

Article Snippet: Astrocytes and brain microvascular endothelial cells were labeled with rabbit anti-glial fibrillary acidic protein (GFAP) (1:100; Boster) and rabbit anti-PECAM-1/CD31 (1:100; Boster) antibodies.

Techniques: Co-Culture Assay, End Labeling, Staining

Journal: Developmental biology

Article Title: Loss of Flow Responsive Tie1 Results in Impaired Aortic Valve Remodeling

doi: 10.1016/j.ydbio.2019.07.011

Figure Lengend Snippet: Key Resource Table (Antibodies for IHC)

Article Snippet: Ultrastructural analysis of endothelial morphology was examined with transmission electron microscopy (EM) as described ( Odelin et al., 2014 ). table ft1 table-wrap mode="anchored" t5 caption a7 Antibodies SOURCE IDENTIFIER Working concentration Rabbit anti-pHH3 Upstate Cat#06–570; PRID:AB_310177 1:100 Rat anti-BrdU Abcam Cat#ab6326; PRID:AB_305426 1:100 Rabbit anti Cleaved Caspase-3 (Asp175) Cell Signaling Cat#9664; PRID:AB_2070042 1:400 Goat anti Tie1 R&D Systems Cat#AF619; PRID:AB_355481 1:50 Goat anti-mouse Tie2 R&D Systems Cat#AF762; PRID:AB_2203220 1:400 Rat anti-mouse CD31 Pharmingen, Cat#557355; PRID:AB_396660 1:200 Mouse anti Collagen III Abcam Cat#ab6310; PRID:AB_305413 1:400 Rabbit anti Collagen I Abcam Cat#ab34710; PRID:AB_731684 1:100 Rabbit anti-Erg1 Abcam Cat#ab92513; PRID:AB_2630401 1:100 Goat anti-VE-Cadherin R&D Systems Cat#AF1002; PRID:AB_2077789 1:400 Rabbit anti-Versican (GAGβ) Chemicon Cat#AB1033; PRID:AB_90462 1:1500 Hyaluronic acid binding protein, biotinylated (HABP) Millipore Cat#385911 1:100 Goat anti-Sox9 Cell Signaling Cat#82630 1:100 Rabbit anti Versican V0,V1 Neo (DPEAAE) ThermoFisher Cat#PA1-1748A; PRID:AB_2304324 1:1500 Sheep anti-Foxc2 R&D Systems Cat#AF6989; PRID:AB_10973139 1:200 NL557 Conjugated donkey anti-sheep IgG R&D Systems Cat#NL010; PRID:AB_884220 1:600 Open in a separate window Key Resource Table (Antibodies for IHC)

Techniques: Concentration Assay, Binding Assay

Journal: Developmental biology

Article Title: Loss of Flow Responsive Tie1 Results in Impaired Aortic Valve Remodeling

doi: 10.1016/j.ydbio.2019.07.011

Figure Lengend Snippet: Polarized expression of Tie1 and other flow sensitive endothelial markers in the remodeling aortic valves. A, Tie1 (red), Cdh5 (red), CD31 (green) and Tie2 (red) expression in remodeling aortic valves was detected by immunostaining Tie1fl/fl hearts at E14.5, E16.5, and E18.5. B and C, AoV sections of P0 Tie1fl/fl heart were co-immunostained for Tie1 (red) and CD31 (green) or Cdh5 (red) and CD31 (green). D, P0 Tie1+/lacz heart AoV sections were stained for (β-Gal, β-galactosidase) with X-gal (blue) and then with Tie2 antibody (red, E). DAPI (blue) denotes nuclei. Scale bars: 50 μm. From E16.5, Tie1 and Cdh5 begin to have higher expression on the arterial sides (arrows); in contrast, CD31 and Tie2 have higher expression on the ventricular sides (arrowheads). F, Quantification of relative expression of Tie1, Cdh5, CD31 and Tie2 in remodeling aortic valves: arterial side (predicted to face oscillatory shear stress) vs. ventricular side (predicted to face high laminar shear stress). *P<0.05, **P<0.0,1***P<0.001 (all compared with E14.5).

Article Snippet: Ultrastructural analysis of endothelial morphology was examined with transmission electron microscopy (EM) as described ( Odelin et al., 2014 ). table ft1 table-wrap mode="anchored" t5 caption a7 Antibodies SOURCE IDENTIFIER Working concentration Rabbit anti-pHH3 Upstate Cat#06–570; PRID:AB_310177 1:100 Rat anti-BrdU Abcam Cat#ab6326; PRID:AB_305426 1:100 Rabbit anti Cleaved Caspase-3 (Asp175) Cell Signaling Cat#9664; PRID:AB_2070042 1:400 Goat anti Tie1 R&D Systems Cat#AF619; PRID:AB_355481 1:50 Goat anti-mouse Tie2 R&D Systems Cat#AF762; PRID:AB_2203220 1:400 Rat anti-mouse CD31 Pharmingen, Cat#557355; PRID:AB_396660 1:200 Mouse anti Collagen III Abcam Cat#ab6310; PRID:AB_305413 1:400 Rabbit anti Collagen I Abcam Cat#ab34710; PRID:AB_731684 1:100 Rabbit anti-Erg1 Abcam Cat#ab92513; PRID:AB_2630401 1:100 Goat anti-VE-Cadherin R&D Systems Cat#AF1002; PRID:AB_2077789 1:400 Rabbit anti-Versican (GAGβ) Chemicon Cat#AB1033; PRID:AB_90462 1:1500 Hyaluronic acid binding protein, biotinylated (HABP) Millipore Cat#385911 1:100 Goat anti-Sox9 Cell Signaling Cat#82630 1:100 Rabbit anti Versican V0,V1 Neo (DPEAAE) ThermoFisher Cat#PA1-1748A; PRID:AB_2304324 1:1500 Sheep anti-Foxc2 R&D Systems Cat#AF6989; PRID:AB_10973139 1:200 NL557 Conjugated donkey anti-sheep IgG R&D Systems Cat#NL010; PRID:AB_884220 1:600 Open in a separate window Key Resource Table (Antibodies for IHC)

Techniques: Expressing, Immunostaining, Staining

Journal: Developmental biology

Article Title: Loss of Flow Responsive Tie1 Results in Impaired Aortic Valve Remodeling

doi: 10.1016/j.ydbio.2019.07.011

Figure Lengend Snippet: Nfatc1enCre mediates loxP excision exclusively in the valvular endocardium. A-D, X-gal staining of R26fslz;Nfatc1enCre heart sections shows that Nfatc1enCre-mediated recombination is restricted to the endothelium of the developing aortic valves (arrows) but not the endothelium of the artery (solid arrowheads), mesenchyme derived from EMT or ventricular endocardium (open arrowheads) at E12.5 (A), at E14.5 (B) and at P0 (C), and lacZ expression remains in the majority of the valvular endothelial cells throughout adulthood (D). E, Representative images of immuno-staining of Tie2-cko AoVs at E14.5 with anti-Tie1 antibody showing efficient specific Tie1 deletion in valvular endothelium (arrows) without change of normal expression of Tie1 in arterial endothelium (solid arrowheads) or ventricular endocardium (open arrowheads). F, Nfatc1enCre-mediated Tie1 deletion was not very efficient in a few (2 out of 9) mutant embryos. G and H, the same sections in E and F were co-stained for CD31, showing the integrity of endothelial cells. Yellow arrows indicate the mosaic Tie1 expression in the mutant valvular endothelium. DAPI, blue. Scale bars: 50μm in A-C, H-I and 200μm in D. I, The efficiency of Cre mediated Tie1 deletion in AoVs was assessed with qRT-PCR on valve leaflets of Tie1fl/fl (control) and Tie1fl/lz;Nfatc1enCre (Tie1-cko) mice at E14.5 and at P0. For normalization, Tie1 mRNA level in control mice at E14.5 was set as 1. ***P<0.001.

Article Snippet: Ultrastructural analysis of endothelial morphology was examined with transmission electron microscopy (EM) as described ( Odelin et al., 2014 ). table ft1 table-wrap mode="anchored" t5 caption a7 Antibodies SOURCE IDENTIFIER Working concentration Rabbit anti-pHH3 Upstate Cat#06–570; PRID:AB_310177 1:100 Rat anti-BrdU Abcam Cat#ab6326; PRID:AB_305426 1:100 Rabbit anti Cleaved Caspase-3 (Asp175) Cell Signaling Cat#9664; PRID:AB_2070042 1:400 Goat anti Tie1 R&D Systems Cat#AF619; PRID:AB_355481 1:50 Goat anti-mouse Tie2 R&D Systems Cat#AF762; PRID:AB_2203220 1:400 Rat anti-mouse CD31 Pharmingen, Cat#557355; PRID:AB_396660 1:200 Mouse anti Collagen III Abcam Cat#ab6310; PRID:AB_305413 1:400 Rabbit anti Collagen I Abcam Cat#ab34710; PRID:AB_731684 1:100 Rabbit anti-Erg1 Abcam Cat#ab92513; PRID:AB_2630401 1:100 Goat anti-VE-Cadherin R&D Systems Cat#AF1002; PRID:AB_2077789 1:400 Rabbit anti-Versican (GAGβ) Chemicon Cat#AB1033; PRID:AB_90462 1:1500 Hyaluronic acid binding protein, biotinylated (HABP) Millipore Cat#385911 1:100 Goat anti-Sox9 Cell Signaling Cat#82630 1:100 Rabbit anti Versican V0,V1 Neo (DPEAAE) ThermoFisher Cat#PA1-1748A; PRID:AB_2304324 1:1500 Sheep anti-Foxc2 R&D Systems Cat#AF6989; PRID:AB_10973139 1:200 NL557 Conjugated donkey anti-sheep IgG R&D Systems Cat#NL010; PRID:AB_884220 1:600 Open in a separate window Key Resource Table (Antibodies for IHC)

Techniques: Staining, Derivative Assay, Expressing, Immunostaining, Mutagenesis, Quantitative RT-PCR

Journal: Stem cells (Dayton, Ohio)

Article Title: Human bone marrow-derived mesenchymal stem cells suppress human glioma growth through inhibition of angiogenesis.

doi: 10.1002/stem.1247

Figure Lengend Snippet: Figure 4. Conditioned medium from DGli36/MSC coculture prevented recruitment of EPC and endothelial tube formation. (A): EPCs were immunostained with antibodies against CD133, CD34, VE-cadherin, VEGFR-2, Tie2, and CD45. Mouse IgG1 and rabbit serum were used as neg- ative control for the first five antibodies and CD45, respectively. Scale bar ¼ 100 lm. (B): EPC and MSC were assessed for their ability to uptake DiI-Ac-LDL (red fluorescence). Cells were counterstained with DAPI (blue fluorescence). Representative images were presented. Scale bar ¼ 50 lm. (C): EPC recruitment assay was performed using DGli36-CM, MSC-CM, and DGli36/MSC-CM. The number of recruited cells to the bottom chamber were counterstained with propidium iodide and counted. Data shown are averages of triplicates 6 SEM. *, p < .05. (D): Angiogenesis assay was performed to determine whether DGli36-CM, MSC-CM, NHA-CM, DGli36/NHA-CM, or DGli36/MSC-CM could induce tube formation of HUVEC. 50 ng/ml of recombinant human VEGF was used as positive control. Tubes formed were visualized and captured using the Zeiss 200M wide-field microscope at 50 original magnification. Scale bar ¼ 200 lm. (E): Tube formation index was scored using ImageJ software (National Institutes of Health, Bethesda, MD). Data were obtained from four replicates of two independent experiments and pre- sented as ratio of coculture to monoculture. Abbreviations: CM, conditioned medium; EPC, endothelial progenitor cells; LDL, low density lipo- protein; NHA, normal human astrocytes; MSC, mesenchymal stem cell; VE, vascular endothelial; VEGFR, vascular endothelial growth factor receptor; DAPI, 40,6-diamidino-2-phenylindole; HUVEC, Human umbilical vein endothelial cells.

Article Snippet: The following antibodies were used for the various staining: CD31 (1 lg/ml; BD Biosciences), cathepsin B (5 lg/ml; R&D Systems, Minneapolis, MN, http://www.rndsystems.com), single-stranded DNA (ssDNA; 1 lg/ml; clone F7-26, Millipore, Temecula, CA, http:// www.millipore.com), a-smooth muscle actin (SMA; 1:100 dilution; Sigma Aldrich, St. Louis, MO, http://www.sigmaaldrich. com), CD34 (1 lg/ml), CD133 (1:100 dilution), Tie2 (1 lg/ml), VE-cadherin (1 lg/ml) and VEGFR-2 (1 lg/ml) (Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com), and CD45 (1:100 dilution; BD Biosciences).

Techniques: Control, Angiogenesis Assay, Recombinant, Positive Control, Microscopy, Software

Journal: Developmental biology

Article Title: Loss of Flow Responsive Tie1 Results in Impaired Aortic Valve Remodeling

doi: 10.1016/j.ydbio.2019.07.011

Figure Lengend Snippet: Key Resource Table (Antibodies for IHC)

Article Snippet: Ultrastructural analysis of endothelial morphology was examined with transmission electron microscopy (EM) as described ( Odelin et al., 2014 ). table ft1 table-wrap mode="anchored" t5 caption a7 Antibodies SOURCE IDENTIFIER Working concentration Rabbit anti-pHH3 Upstate Cat#06–570; PRID:AB_310177 1:100 Rat anti-BrdU Abcam Cat#ab6326; PRID:AB_305426 1:100 Rabbit anti Cleaved Caspase-3 (Asp175) Cell Signaling Cat#9664; PRID:AB_2070042 1:400 Goat anti Tie1 R&D Systems Cat#AF619; PRID:AB_355481 1:50 Goat anti-mouse Tie2 R&D Systems Cat#AF762; PRID:AB_2203220 1:400 Rat anti-mouse CD31 Pharmingen, Cat#557355; PRID:AB_396660 1:200 Mouse anti Collagen III Abcam Cat#ab6310; PRID:AB_305413 1:400 Rabbit anti Collagen I Abcam Cat#ab34710; PRID:AB_731684 1:100 Rabbit anti-Erg1 Abcam Cat#ab92513; PRID:AB_2630401 1:100 Goat anti-VE-Cadherin R&D Systems Cat#AF1002; PRID:AB_2077789 1:400 Rabbit anti-Versican (GAGβ) Chemicon Cat#AB1033; PRID:AB_90462 1:1500 Hyaluronic acid binding protein, biotinylated (HABP) Millipore Cat#385911 1:100 Goat anti-Sox9 Cell Signaling Cat#82630 1:100 Rabbit anti Versican V0,V1 Neo (DPEAAE) ThermoFisher Cat#PA1-1748A; PRID:AB_2304324 1:1500 Sheep anti-Foxc2 R&D Systems Cat#AF6989; PRID:AB_10973139 1:200 NL557 Conjugated donkey anti-sheep IgG R&D Systems Cat#NL010; PRID:AB_884220 1:600 Open in a separate window Key Resource Table (Antibodies for IHC)

Techniques: Concentration Assay, Binding Assay