Mouse DLL4 Antibody

Detection of Mouse DLL4 by Western Blot. Western blot shows lysates of bEnd.3 mouse endothelioma cell line. PVDF membrane was probed with 2 µg/mL of Goat Anti-Mouse DLL4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1389) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). A specific band was detected for DLL4 at approximately 90 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

DLL4 in bEnd.3 Mouse Cell Line. DLL4 was detected in immersion fixed bEnd.3 mouse endothelioma cell line using Goat Anti-Mouse DLL4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1389) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; Catalog # NL001) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

DLL4 in Mouse Embryo. DLL4 was detected in immersion fixed paraffin-embedded sections of mouse embryo (13 d.p.c.) using Goat Anti-Mouse DLL4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1389) at 5 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Goat IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC004). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to developing vasculature. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Detection of Mouse DLL4 by Western Blot MPDZ promotes Notch signaling activity.(A) HUVECs were either transduced with lentivirus expressing GFP (sh-ctrl) or with lentivirus expressing shRNA against MPDZ (sh-MPDZ). Expression level of Notch target genes HEY1, HEY2 and HES1 were analyzed by qPCR 48 hr after transduction. Data are presented as mean ±SD. n ≥ 3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student’s t-test. (B) Cardiac endothelial cells were isolated from Mpdzfl/fl and Mpdz delta EC mice by magnetic beads bound with CD31 antibodies. Expression levels of Notch target genes Hey1 and Hey2 were analyzed by qPCR. Data are presented as mean ±SD. n = 3; *, p<0.05; ***, p<0.001 unpaired Student’s t-test. (C) HUVECs were either transduced with lentivirus expressing GFP (sh-ctrl) or with lentivirus expressing shRNA against MPDZ (sh-MPDZ). Expression levels of DLL1 and DLL4 were analyzed by immunoblotting 48 hr after transduction. beta -actin served as loading control. Data are presented as mean ±SD. n ≥ 3; n.s., not significant. (D) HUVECs were either transduced with adenovirus expressing GFP (ctrl) or with adenovirus expressing MPDZ. Expression levels of DLL1 and DLL4 were analyzed by immunoblotting 48 hr after transduction. beta -actin served as loading control. Data are presented as mean ±SD. n ≥ 3; n.s., not significant. (E) Lung endothelial cells were isolated from Mpdzfl/fl and Mpdz delta EC mice by CD31 magnetic beads. Protein amounts of Dll1 and Dll4 were analyzed by immunoblotting. beta -actin served as loading control. Data are presented as mean ±SD. n = 3; n.s., not significant.10.7554/eLife.32860.007Figure 2—source data 1.Source data of qantitative PCR analysis related to Figure 2A and B.Source data of qantitative PCR analysis related to Figure 2A and B. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/29620522), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse DLL4 by Immunocytochemistry/ Immunofluorescence Mpdz does not affect cell cell junction assembly.(A, B) HUVECs were either transduced with lentivirus expressing GFP (sh-ctrl) or with lentivirus expressing shRNA against MPDZ (sh-MPDZ). Cells were cultured under sparse conditions (A) or confluent conditions (B). After PFA fixation cells were stained for DLL1 and Nectin-2 or DLL4 and Nectin-2 and counterstained with DAPI. Images were acquired with the confocal microscope LSM 700. Arrow indicates co-localization of DLL1/4 with Nectin-2 at the cell membrane. Arrow head indicates diminished co-localization at the cell membrane. Scale bar: 10 µm. (C) HUVECs were either transfected with control siRNA (si-ctrl) or with siRNA against Nectin-2 (si-Nectin-2). After PFA fixation cells were stained for DLL1 and Nectin-2 or DLL4 and Nectin-2. Images were acquired with the confocal microscope LSM 700. Arrow indicates localization of DLL1/4 at the cell membrane.Scale bar: 10 µm. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/29620522), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse DLL4 by Western Blot MPDZ promotes Notch signaling activity.(A) HUVECs were either transduced with lentivirus expressing GFP (sh-ctrl) or with lentivirus expressing shRNA against MPDZ (sh-MPDZ). Expression level of Notch target genes HEY1, HEY2 and HES1 were analyzed by qPCR 48 hr after transduction. Data are presented as mean ±SD. n ≥ 3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student’s t-test. (B) Cardiac endothelial cells were isolated from Mpdzfl/fl and Mpdz delta EC mice by magnetic beads bound with CD31 antibodies. Expression levels of Notch target genes Hey1 and Hey2 were analyzed by qPCR. Data are presented as mean ±SD. n = 3; *, p<0.05; ***, p<0.001 unpaired Student’s t-test. (C) HUVECs were either transduced with lentivirus expressing GFP (sh-ctrl) or with lentivirus expressing shRNA against MPDZ (sh-MPDZ). Expression levels of DLL1 and DLL4 were analyzed by immunoblotting 48 hr after transduction. beta -actin served as loading control. Data are presented as mean ±SD. n ≥ 3; n.s., not significant. (D) HUVECs were either transduced with adenovirus expressing GFP (ctrl) or with adenovirus expressing MPDZ. Expression levels of DLL1 and DLL4 were analyzed by immunoblotting 48 hr after transduction. beta -actin served as loading control. Data are presented as mean ±SD. n ≥ 3; n.s., not significant. (E) Lung endothelial cells were isolated from Mpdzfl/fl and Mpdz delta EC mice by CD31 magnetic beads. Protein amounts of Dll1 and Dll4 were analyzed by immunoblotting. beta -actin served as loading control. Data are presented as mean ±SD. n = 3; n.s., not significant.10.7554/eLife.32860.007Figure 2—source data 1.Source data of qantitative PCR analysis related to Figure 2A and B.Source data of qantitative PCR analysis related to Figure 2A and B. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/29620522), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse DLL4 by Immunocytochemistry/ Immunofluorescence Endothelial changes after pericyte depletion. a–f Maximum intensity projection of confocal images from control and DTRiPC P6 retinas stained for IB4 (red) in combination with VEGF-A a, VEGFR2 b, VEGFR3 c, Tie2 d, Esm1 e, and Dll4 f (all in white), as indicated. Note local increase of VEGFR2, VEGFR3, and Esm1 (arrowheads in b, c, e) but not Tie2 or VEGF-A at the edge of the vessel plexus. Dll4 expression in DTRiPC sprouts is increased in some regions (arrowheads) but absent in others (arrows). Scale bar, 100 µm. g–j Quantitation of VEGF-A immunosignals area and intensity g, signal intensity for VEGFR2 h and VEGFR3 i and proportion of Esm1+ area with respect to vascular area j in the P6 control and DTRiPC angiogenic front. Error bars, s.e.m. p-values, Student’s t-test. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/29146905), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse DLL4 by Immunohistochemistry Targeting of Fbxw7 induced the upregulation of Dll4 expression.Anti-Dll4 and Isolectin B4 (IsolB4)-stained retinal whole-mounts of Fbxw7iECKO and littermate control retinas, as indicated (A, B). Arrowheads in (B) mark Dll4+ peripheral sprouts at the edge of the growing plexus, arrows indicate upregulated Dll4 in Fbxw7iECKO retinal capillaries. Quantitative analysis (with Volocity 5; n = 3 for each group) of image data confirmed elevated Dll4 levels (number of pixels) in the mutant endothelium (C). Likewise, quantitative RT-PCR analysis showed reduced Fbxw7 expression but upregulated Dll4 transcript levels in P6 Fbxw7iECKO lungs (D). Expression of the Cdh5 gene was used for normalization. Error bars indicate SEM. P values are indicated as ** (<0.001) and * (p<0.05). Scale bars are 100 µm. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/22848434), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse DLL4 by Immunohistochemistry Targeting of Fbxw7 induced the upregulation of Dll4 expression.Anti-Dll4 and Isolectin B4 (IsolB4)-stained retinal whole-mounts of Fbxw7iECKO and littermate control retinas, as indicated (A, B). Arrowheads in (B) mark Dll4+ peripheral sprouts at the edge of the growing plexus, arrows indicate upregulated Dll4 in Fbxw7iECKO retinal capillaries. Quantitative analysis (with Volocity 5; n = 3 for each group) of image data confirmed elevated Dll4 levels (number of pixels) in the mutant endothelium (C). Likewise, quantitative RT-PCR analysis showed reduced Fbxw7 expression but upregulated Dll4 transcript levels in P6 Fbxw7iECKO lungs (D). Expression of the Cdh5 gene was used for normalization. Error bars indicate SEM. P values are indicated as ** (<0.001) and * (p<0.05). Scale bars are 100 µm. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/22848434), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Mouse DLL4 Antibody by Immunohistochemistry-Frozen DLL4 expressed from the Dll1 locus rescues DLL1 loss-of-function in the retina.(A)Dll1 null mutant retinas show epithelial disruption with formation of polarised rosettes in which the apical markers N-Cadherin (NCad, a) and ZO-1 (ZO1, b) are abnormally present at the central lumen. Ectopic proliferating progenitors, labelled with PHH3 (b, arrowheads), are located close to the apical lumen of these rosettes. (B) In contrast, the neuroepithelium of homozygous Dll1Dll1ki and Dll1Dll4ki embryos is correctly organised without rosettes, and N-Cadherin shows the normal apical localisation close to the retinal pigmented epithelium (a,b). Mitotic progenitors (PHH3+) are only detected at the apical region of the neuroepithelium (a,b arrowheads). A normal stratification of CHX10+ progenitors and P27+ differentiating neurons is also observed (c,d). (C, D) E13.5 homozygous Dll1Dll1ki and Dll1Dll4ki retinas show no significant difference in the number of ISL1+ RGCs (C) and CRABP+ amacrine cells (D). Cells immunopositive for Islet-1 and Crabp were counted and related to the total number of cells in the retina (DAPI+). Percentages are shown as mean ± SEM; ns, not significant. (E) Expression of DLL4 in homozygous Dll1Dll1ki (a,c) and in homozygous Dll1Dll4ki (b,d) E13.5 retinas as detected by an anti-DLL4 antibody. (c) and (d) are magnifications of (a) and (b), respectively. Endogenous plus transgenic DLL4 is expressed in more cells in Dll1Dll4ki/Dll4ki as compared to endogenous DLL4 expression in Dll1Dll1ki/Dll1ki while signal strength is similar. Scale bars are 50 μm in (A, B) and 100 μm in (E). Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/26114479), licensed under a CC-BY license. Not internally tested by R&D Systems.