Mouse Endomucin Antibody

Detection of Mouse Endomucin by Western Blot. Western blot shows lysates of mouse kidney tissue, mouse lung tissue, and mouse ovary tissue. PVDF membrane was probed with 1 µg/mL of Goat Anti-Mouse Endomucin Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4666) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). A specific band was detected for Endomucin at approximately 75-85 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Endomucin in bEnd.3 Mouse Cell Line. Endomucin was detected in immersion fixed bEnd.3 mouse endothelioma cell line using Goat Anti-Mouse Endomucin Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4666) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 493-conjugated Anti-Goat IgG Secondary Antibody (green; Catalog # NL003) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Detection of Endomucin in bEnd.3 Mouse Cell Line by Flow Cytometry. bEnd.3 mouse endothelioma cell line was stained with Goat Anti-Mouse Endomucin Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4666, filled histogram) or isotype control antibody (Catalog # AB-108-C, open histogram), followed by Allophycocyanin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0108).

Detection of Mouse Endomucin/Mucin -14 by Immunocytochemistry/Immunofluorescence MLK2/3-deficient mice exhibit defects in native collateral artery formation.(A) Representative confocal images (n = 5 mice) of control and MLK2/3-deficient whole mount P6 adductor muscle vasculature stained with antibodies to endomucin (capillary and venous vasculature, red) and SMA (arterial and venous smooth muscle, green). Gracilis collateral arteries in WT mice, but not MLK2/3-deficient mice, interconnect the PCFA to the SA. (B) Representative stereomicroscope images of P6 whole mount abdominal muscle stained with an antibody to SMA (green). Arteriole-to-arteriole arcades are indicated (red arrows). The abdominal muscle vasculature of Map3k10-/-Map3k11-/- mice shows very few arteriole-to-arteriole interconnections. Quantitation reveals significantly reduced arteriolar arcade numbers in Map3k10-/-Map3k11-/- mice compared to WT mice (mean ± SEM; n = 5 mice). Source data are included as Figure 3—source data 1.DOI:https://dx.doi.org/10.7554/eLife.18414.02310.7554/eLife.18414.024Figure 3—source data 1.Source data for Figure 3.This file contains raw source data used to make the graphs presented in Figure 3.DOI:https://dx.doi.org/10.7554/eLife.18414.024Source data for Figure 3.This file contains raw source data used to make the graphs presented in Figure 3.DOI:https://dx.doi.org/10.7554/eLife.18414.024 Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/27504807), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Endomucin/Mucin -14 by Immunocytochemistry/Immunofluorescence NOX2 contributes to the oxidative stress of bone ECs and the damage of angiogenesis-osteogenesis coupling. (A) qPCR analysis of the relative mRNA levels of three critical NOXs, involved in endothelial oxidative injuries, in the ECs in tibia metaphysis. T1DM mice were intraperitoneally injected with GKT137831 (NOX1/4 inhibitor) or one of the two NOX2 inhibitors, NOX2ds-tat (NOX2ds) and GSK2795039 (GSK), or VAS2870 (VAS, a pan-NOX inhibitor) once every two days from week 4 through 8. The levels of blood glucose (B), insulin (C) and body weight (D) during treatments were tested. (E) Confocal images of immunostained tibia sections show the CD31+Emcn+ type H vessels in the metaphysis at week 8. Scale bar: 50 μm on the above and 20 μm on the below. (F and G) Histomorphometric quantitation of the vessels in proximal tibia metaphysis. Flow cytometry quantification of type H ECs and total ECs in 8-week-old tibia (H). (I) Flow cytometry analysis of intracellular DCF fluorescence shows the ROS levels in the ECs in 8-week-old tibia. (B)-(D), n = 15-16 per group; other panels, n = 7-8 per group. ###p < 0.001 vs. Control group; *p < 0.05, **p < 0.01 and ***p < 0.001 vs. DM+veh group. One-way or two-way ANOVA followed by Tukey posttest analysis. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/33664862), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Endomucin/Mucin -14 by Immunocytochemistry/Immunofluorescence The effects of anti-oxidant treatments on the bone blood vessels of T1DM mice. T1D mice received intraperitoneal injection of an anti-oxidant, N-acetyl cysteine (NAC) or tempol (TPO), once every other day from week 4 through 8. (A) Flow cytometry analysis of the general ROS levels in the bone ECs from 8-week-old tibia by detecting intracellular DCF fluorescence. The levels of blood glucose (B), insulin (C) and body weight (D). (E) Tibia length at week 8. (F) Confocal images of CD31 (green) and Emcn (red) immunostained tibia sections show type H vessels in the tibia metaphysis at week 8. Scale bar: 50 μm on the above and 20 μm on the below. Histomorphometric quantitation of the area fraction of vessel columns (G) and the number of vascular buds per growth plate length (H). Flow cytometry quantification of type H ECs (I) and total ECs (J) in 8-week-old tibia. (K) qPCR analysis of the representative cytokines mediating the coupling of angiogenesis and osteogenesis in 8-week-old tibia. (B)-(D), n = 15-16 per group; other panels, n = 7-8 per group. *p < 0.05, **p < 0.01 and ***p < 0.001. One-way or two-way ANOVA followed by Tukey posttest analysis. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/33664862), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Endomucin/Mucin -14 by Immunocytochemistry/Immunofluorescence Endothelial JNK-deficient mice display abnormal native collateral arteries.(A,B) Representative confocal images (n = 7 mice) of whole mount adductor muscle vasculature reveals SMA-covered gracilis collateral arteries in P6 control mice, but not JNK-deficient mice (A). Confocal imaging of Dil perfused P6 adductor muscle vasculature (n = 5 mice) demonstrates distinct gracilis collaterals interconnecting the PCFA to the SA in control mice. Vessels emerging from the PCFA and the SA in E3KO mice do not fully interconnect, but branch into smaller vessels. At P0, gracilis collaterals were not SMA-covered, but were perfused with Dil in control mice (B). The analogous vessels in E3KO mice did not interconnect, but branched extensively into smaller vessels (B) (n = 5 mice). (C) Representative stereomicroscope images of Dil-perfused abdominal muscle arterial vasculature of control and JNK-deficient P0 mice. Arteriole-to-arteriole arcades (indicated by red arrows) were quantitated (mean ± SEM; n = 3 mice). (D) Representative confocal images (n = 3~4 mice) of whole mount adductor muscles of control and JNK-deficient E16.5 embryos showing GFP-labeled vascular endothelial cells. (E) Representative confocal images (n = 3~5 mice) of control and JNK-deficient E16.5 embryo adductor muscle vasculature immunostained for Endomucin (Emcn, red) and isolectinB4 (iB4, green). Prominent vessels emerging from the PCFA and SA are indicated with white arrowheads. Source data are included as Figure 5—source data 1.DOI:https://dx.doi.org/10.7554/eLife.18414.02710.7554/eLife.18414.028Figure 5—source data 1.Source data for Figure 5.This file contains raw source data used to make the graphs presented in Figure 5.DOI:https://dx.doi.org/10.7554/eLife.18414.028Source data for Figure 5.This file contains raw source data used to make the graphs presented in Figure 5.DOI:https://dx.doi.org/10.7554/eLife.18414.028Intimate association of gracilis collaterals and peripheral nerves in adductor muscles.Confocal microscopy of a whole mount adductor muscle stained with antibodies to SMA (green) and Neurofilament-M (red) illustrates the close association of gracilis collateral arteries with peripheral nerves.DOI:https://dx.doi.org/10.7554/eLife.18414.029 Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/27504807), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Endomucin/Mucin -14 by Immunocytochemistry/Immunofluorescence The effects of insulin therapy on the bone blood vessels of T1DM mice. Drug therapy with high dose (hi, 0.5 U/mouse) or low dose (lo, 0.05 U/mouse) of insulin (ins) or the combination of insulin and a relatively low dose of GSK2795039 (GSK, a NOX2 inhibitor, 2 mM) was conducted in two time courses: started immediately after the confirmation of DM and once per day in week 4 to 8 (4-8w); started at week 6 and performed once per day in week 6 to 8 (6-8w). The levels of blood glucose (A) and body weight (B) were examined at week 4, 6 and 8, with HbA1c (C) tested at week 8. (D) Confocal images of immunostained tibia sections show CD31+ (green) and Emcn+ (red) type H vessels in the metaphysis. Scale bar: 50 μm on the above and 20 μm on the below. Histomorphometric quantitation as the area fraction of vessel columns (E) and the number of vascular buds per growth plate length (F). (G) Flow cytometry quantification of type H ECs and total ECs in 8-week-old tibia. For drug treatments, T1DM model was induced by STZ injection at 80 mg/kg (namely DM-G2). (A)-(C), n = 15-16 per group; (E)-(G), n = 6-8 per group. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. DM+veh group; #p < 0.05, ##p < 0.01 and ###p < 0.001 vs. DM+ins hi 6-8w group; &p < 0.05, &&p < 0.01 and &&&p < 0.001 vs. DM+ins lo 4-8w group. One-way or two-way ANOVA followed by Tukey posttest analysis. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/33664862), licensed under a CC-BY license. Not internally tested by R&D Systems.