iowa ab 528222 rat anti mouse anti cd105 endoglin  (Developmental Studies Hybridoma Bank)

Journal: The European journal of neuroscience

Article Title: VEGF-E Attenuates Injury After Ischemic Stroke by Promoting Reparative Revascularization.

doi: 10.1111/ejn.70114

Figure Lengend Snippet: FIGURE 3 | Revascularization is stabilized by VEGF-E associated with attenuation of neuronal degeneration. (a) Representative fluorescence images of CD31 and PDGFRβ immunolabeling at the injury site in vehicle (VEH)- and VEGF-E-treated mice 4 days after stroke. (b) Analysis of the density of perivascular PDGFRβ+ cells at the injury site 4 days after stroke. (c) Analysis of CD31 and PDGFRβ colocalization at the injury site. (d) Analysis of the number of CD31+ microvascular stalls at the injury site. (e) Representative fluorescence images of CD13 and CD105 immunolabeling at the injury site in VEH- and VEGF-E-treated mice. (f) Analysis of the density of CD105+ microvessels at the injury site. (g) Analysis of CD105 and CD13 colocalization at the injury site. (h) Representative fluorescence images of FJB+ degenerating neurons in the ipsilateral cortex and ipsilateral striatum. Stereological analysis of the density of FJB+ degenerating neurons in the (i) ipsilateral cortex and (j) ipsilateral striatum. Data are boxplot with min/max or stacked histogram (n = 6–7 animals/group). *p < 0.05, **p < 0.01, ***p < 0.001 compared to control (b, d, f, i, unpaired two-tailed t- test). Abbreviations: IF, immunofluorescence; VEGF, vascular endothelial growth factor; VEH, vehicle.

Article Snippet: The following primary antibodies were used: rat anti- mouse cluster of differentiation (CD31) (1/500; BD Biosciences, ON, Canada; 550274), goat anti- mouse aminopeptidase N (CD13) (1/250; R&D systems), rat anti- mouse glial fibrillary acidic protein (GFAP) (1/500; Invitrogen, MA, USA; 13- 0300), rabbit anti- mouse ionized calcium binding adaptor molecule (IBA1) (1/500; WAKO, ON, Canada; 019- 19741), goat anti- rat CD45 (1/500; BD Biosciences; 553076), rabbit anti- mouse PDGFRβ (1/250; Abcam, ON, Canada; ab32570), rat anti- mouse endoglin (CD105) (1/250; Novus Biologicals, ON, Canada; NB100- 77666), and rabbit anti- mouse aquaporine- 4 (AQP4) (1/500; Cell Signaling Technology, MA, USA; 59678S).

Techniques: Fluorescence, Immunolabeling, Control, Two Tailed Test, Immunofluorescence

Journal: Theranostics

Article Title: Exosome-mediated delivery of an anti-angiogenic peptide inhibits pathological retinal angiogenesis

doi: 10.7150/thno.54755

Figure Lengend Snippet: EXO KV11 suppresses VEGF-induced vascular leakage in vivo . (A) Schematic depiction of the pre-treatment procedure in a VEGF-induced vascular leakage model. EXO KV11 , KV11, EXO, or vehicle was retro-orbitally injected in adult wild type C57BL/6J mice. After 24 h, 100 ng VEGF was intravitreally injected to induce vascular leakage in the retina. The mice were sacrificed for analysis after another 24 h. (B) FITC-dextran was injected and the retinas were harvested at the endpoint. Representative images of flat-mounted retina show extravasated FITC-dextran and CD105 + vessels. (C) Quantification of dextran leakage in (B) (n = 7 - 10 mice per condition). (D) Representative confocal images of anti-CD105, anti-Claudin-5-stained retinal vessels in mice treated as in (A). (E) Representative images of F4/80 + macrophages (green) and CD105 + vessels in retinas treated as in (A). (F) Quantification of macrophage infiltration in (E) (n = 4 - 9 mice per condition). (G) Representative photographs of Evans blue leakage in SD rats ears in an auricular Miles assay with intradermal injection with VEGF, VEGF+KV11 mixture, or VEGF+EXO KV11 mixture. The data represent as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA followed by Tukey's multiple comparisons test in (C), (F). Scale bars, 50 µm.

Article Snippet: For IF staining of red blood cells, blood vessels, macrophage and microglia, flat-mounted retinas were incubated in blocking solution with following primary antibodies at 4 °C overnight: rat anti-TER119 monoclonal (1:200, MAB1125, RD, US); rat anti-CD105 monoclonal (1:200, MAB1320, RD, US); rabbit anti-F4/80 monoclonal (1:200, 30325S, Cell Signaling Technology, US); mouse anti-Claudin-5 monoclonal (1:200, 35-2500, Invitrogen, US) and rabbit anti-ZO-1 polyclonal (1:300, 61-7300, Invitrogen, US).

Techniques: In Vivo, Injection, Staining

Journal: Theranostics

Article Title: Exosome-mediated delivery of an anti-angiogenic peptide inhibits pathological retinal angiogenesis

doi: 10.7150/thno.54755

Figure Lengend Snippet: EXO KV11 has therapeutic effect in post-treatment model. (A) Schematic depiction of the post-treatment procedure in VEGF-induced vascular leakage model. 100 ng VEGF was intravitreally injected to induce vascular leakage in the retina. After 24 h, EXO KV11 , KV11, EXO, or vehicle was retro-orbitally injected in adult wild type C57BL/6J mice. The mice were sacrificed for analysis after another 48 h. (B) FITC-dextran was injected and the retinas were harvested after treatment. Representative images of flat-mounted retina show extravasated FITC-dextran and CD105 + vessels. (C) Quantification of dextran leakage in (B) (n = 4 - 6 mice per condition). (D) Representative images of F4/80 + macrophages (green) and CD105 + vessels in retinas treated as in (A). (E) Quantification of macrophage infiltration in (D) (n = 4 - 6 mice per condition). The data represent as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA followed by Tukey's multiple comparisons test in (C), (E). Scale bars, 50 µm.

Article Snippet: For IF staining of red blood cells, blood vessels, macrophage and microglia, flat-mounted retinas were incubated in blocking solution with following primary antibodies at 4 °C overnight: rat anti-TER119 monoclonal (1:200, MAB1125, RD, US); rat anti-CD105 monoclonal (1:200, MAB1320, RD, US); rabbit anti-F4/80 monoclonal (1:200, 30325S, Cell Signaling Technology, US); mouse anti-Claudin-5 monoclonal (1:200, 35-2500, Invitrogen, US) and rabbit anti-ZO-1 polyclonal (1:300, 61-7300, Invitrogen, US).

Techniques: Injection

Journal: Theranostics

Article Title: Exosome-mediated delivery of an anti-angiogenic peptide inhibits pathological retinal angiogenesis

doi: 10.7150/thno.54755

Figure Lengend Snippet: EXO KV11 suppresses VEGF-induced vascular leakage in vivo . (A) Schematic depiction of the pre-treatment procedure in a VEGF-induced vascular leakage model. EXO KV11 , KV11, EXO, or vehicle was retro-orbitally injected in adult wild type C57BL/6J mice. After 24 h, 100 ng VEGF was intravitreally injected to induce vascular leakage in the retina. The mice were sacrificed for analysis after another 24 h. (B) FITC-dextran was injected and the retinas were harvested at the endpoint. Representative images of flat-mounted retina show extravasated FITC-dextran and CD105 + vessels. (C) Quantification of dextran leakage in (B) (n = 7 - 10 mice per condition). (D) Representative confocal images of anti-CD105, anti-Claudin-5-stained retinal vessels in mice treated as in (A). (E) Representative images of F4/80 + macrophages (green) and CD105 + vessels in retinas treated as in (A). (F) Quantification of macrophage infiltration in (E) (n = 4 - 9 mice per condition). (G) Representative photographs of Evans blue leakage in SD rats ears in an auricular Miles assay with intradermal injection with VEGF, VEGF+KV11 mixture, or VEGF+EXO KV11 mixture. The data represent as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA followed by Tukey's multiple comparisons test in (C), (F). Scale bars, 50 µm.

Article Snippet: In VEGF induced retina vascular leakage model, retinas were incubated in blocking solution with the primary antibody rat anti-CD105 monoclonal (1:200, MAB1320, RD, US) at 4 °C overnight after permeabilization and blocking.

Techniques: In Vivo, Injection, Staining

Journal: Theranostics

Article Title: Exosome-mediated delivery of an anti-angiogenic peptide inhibits pathological retinal angiogenesis

doi: 10.7150/thno.54755

Figure Lengend Snippet: EXO KV11 has therapeutic effect in post-treatment model. (A) Schematic depiction of the post-treatment procedure in VEGF-induced vascular leakage model. 100 ng VEGF was intravitreally injected to induce vascular leakage in the retina. After 24 h, EXO KV11 , KV11, EXO, or vehicle was retro-orbitally injected in adult wild type C57BL/6J mice. The mice were sacrificed for analysis after another 48 h. (B) FITC-dextran was injected and the retinas were harvested after treatment. Representative images of flat-mounted retina show extravasated FITC-dextran and CD105 + vessels. (C) Quantification of dextran leakage in (B) (n = 4 - 6 mice per condition). (D) Representative images of F4/80 + macrophages (green) and CD105 + vessels in retinas treated as in (A). (E) Quantification of macrophage infiltration in (D) (n = 4 - 6 mice per condition). The data represent as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA followed by Tukey's multiple comparisons test in (C), (E). Scale bars, 50 µm.

Article Snippet: In VEGF induced retina vascular leakage model, retinas were incubated in blocking solution with the primary antibody rat anti-CD105 monoclonal (1:200, MAB1320, RD, US) at 4 °C overnight after permeabilization and blocking.

Techniques: Injection

Journal: International Journal of Molecular Sciences

Article Title: Thresholds of Endoglin Expression in Endothelial Cells Explains Vascular Etiology in Hereditary Hemorrhagic Telangiectasia Type 1

doi: 10.3390/ijms22168948

Figure Lengend Snippet: Endoglin mRNA expression levels in multiple mouse organs. ( A ) Ct values for all candidate genes in skin, lung, intestine, brain, heart, kidney and liver isolated from 9 weeks old 129/Svj ( n = 6) and C57Bl/6J ( n = 7) mice. ( B ) Average expression stability values (M) of the candidate reference. M is represented from the least stable (left) to the most stable (right), analyzed by GeNorm software. ( C ) Pairwise variation analysis between normalization factors to determine the optimal number of control genes for normalization. ( D , E ) Real time PCR for endoglin normalized to a reference pool of three endothelial reference genes ( Tie1, Tie2 and Icam2 ) determined by GeNorm comparing endoglin levels in multiple tissues isolated from 9 weeks old 129/Svj ( n = 6) and C57Bl/6J ( n = 7) mice. ( D ) Non significant (ns) results from Mann–Whitney U test that compares the median of two groups. ( E ) Error bars represent SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 result from one-way ANOVA and Tukey’s multiple comparison tests comparing the mean of each group with the mean of the other groups. ns: Not significant.

Article Snippet: The ear microvasculature was labelled with rat anti-mouse Pecam1 (Clone 13.3) (550274, BD Pharmigen, Franklin Lake, NJ, USA) or with rat anti-mouse endoglin (clone 2Q1707) (sc-71042, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and then incubated with Alexa Fluor ® 555 donkey anti-rat (A-21434, Invitrogen, Waltham, MA, USA) and with monoclonal anti-α-smooth muscle actin conjugated with FITC (clone 1A4) (F3777, Sigma-Aldrich, Saint louis, MO, USA).

Techniques: Expressing, Isolation, Software, Control, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Thresholds of Endoglin Expression in Endothelial Cells Explains Vascular Etiology in Hereditary Hemorrhagic Telangiectasia Type 1

doi: 10.3390/ijms22168948

Figure Lengend Snippet: Marked heterogeneity in endoglin levels across different mouse tissues. ( A , B ) Sections of multiple organs isolated from 9-week-old C57Bl/6J mice stained for Pecam1 ( A ) or endoglin ( B ) (scale bar: 50 μm). ( C ) Fluorescence intensity was measured over the full range of PMT voltages for liver and the skin sections to determine the linear fluorescence intensities for each tissue. We identified the PMT gain of 850 as optimal to ensure an accurate quantification of endoglin levels across the various tissues. ( D ) Endoglin levels were quantified using ImageJ software defined as the mean grey intensity of the blood capillaries selected in each image. A minimum of 30 blood vessels was quantified per organ isolated from 3 wild-type mice. Error bars represent SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 result from 1-way ANOVA and Tukey’s multiple comparison tests comparing the mean of each group with the mean of the other groups. ns: Not significant.

Article Snippet: The ear microvasculature was labelled with rat anti-mouse Pecam1 (Clone 13.3) (550274, BD Pharmigen, Franklin Lake, NJ, USA) or with rat anti-mouse endoglin (clone 2Q1707) (sc-71042, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and then incubated with Alexa Fluor ® 555 donkey anti-rat (A-21434, Invitrogen, Waltham, MA, USA) and with monoclonal anti-α-smooth muscle actin conjugated with FITC (clone 1A4) (F3777, Sigma-Aldrich, Saint louis, MO, USA).

Techniques: Isolation, Staining, Fluorescence, Software, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Thresholds of Endoglin Expression in Endothelial Cells Explains Vascular Etiology in Hereditary Hemorrhagic Telangiectasia Type 1

doi: 10.3390/ijms22168948

Figure Lengend Snippet: Heterogeneity in endoglin expression along the arteriovenous axis. ( A ) Fluorescence intensity was measured over the full range of PMT voltages for liver, lung and skin sections from 9-week-old C57Bl/6J wild type ( n = 3) and Eng +/− mice ( n = 3) and confirmed that endoglin levels were reduced by half in all organs. ( B ) Upper panels. Confocal images of whole mount ears isolated from 8-week-old wild-type and Eng +/− mice and stained for endoglin or PECAM1 and α-SMA (Scale bar: 500 μm). Lower panels are higher magnifications showing that endoglin is predominantly expressed in veins and becomes almost undetectable in arteries and capillaries in Eng +/− mice (Scale bar: 100 μm). Error bars represent SD. * p < 0.05, ** p < 0.01 and **** p < 0.0001 result from 2-way ANOVA and Šidàk’s multiple comparisons test.

Article Snippet: The ear microvasculature was labelled with rat anti-mouse Pecam1 (Clone 13.3) (550274, BD Pharmigen, Franklin Lake, NJ, USA) or with rat anti-mouse endoglin (clone 2Q1707) (sc-71042, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and then incubated with Alexa Fluor ® 555 donkey anti-rat (A-21434, Invitrogen, Waltham, MA, USA) and with monoclonal anti-α-smooth muscle actin conjugated with FITC (clone 1A4) (F3777, Sigma-Aldrich, Saint louis, MO, USA).

Techniques: Expressing, Fluorescence, Isolation, Staining

Journal: International Journal of Molecular Sciences

Article Title: Thresholds of Endoglin Expression in Endothelial Cells Explains Vascular Etiology in Hereditary Hemorrhagic Telangiectasia Type 1

doi: 10.3390/ijms22168948

Figure Lengend Snippet: Endoglin expression levels in human tissues. ( A , B ) Sections of multiple tissues isolated from a 57 year-old patient with HHT1 stained for PECAM1 ( A ) or endoglin ( B ) (Scale bar: 50 μm). Pecam1 and endoglin levels were quantified using ImageJ software defined as the mean grey intensity of the blood capillaries selected in each image. Blood vessels were quantified per organ isolated from at least three independent tissue sections. Error bars represent SEM. ** p < 0.01, *** p < 0.001 and **** p < 0.0001 result from 1-way ANOVA and Tukey’s multiple comparison tests comparing the mean of each group with the mean of the other groups. ns: Not significant.

Article Snippet: The ear microvasculature was labelled with rat anti-mouse Pecam1 (Clone 13.3) (550274, BD Pharmigen, Franklin Lake, NJ, USA) or with rat anti-mouse endoglin (clone 2Q1707) (sc-71042, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and then incubated with Alexa Fluor ® 555 donkey anti-rat (A-21434, Invitrogen, Waltham, MA, USA) and with monoclonal anti-α-smooth muscle actin conjugated with FITC (clone 1A4) (F3777, Sigma-Aldrich, Saint louis, MO, USA).

Techniques: Expressing, Isolation, Staining, Software, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Thresholds of Endoglin Expression in Endothelial Cells Explains Vascular Etiology in Hereditary Hemorrhagic Telangiectasia Type 1

doi: 10.3390/ijms22168948

Figure Lengend Snippet: Different sensitivity of organ-specific ECs to reduced endoglin levels. ( A ) Schematic illustration of the strategy used to study how endoglin levels influence EC signaling pathways. It includes the microbeads-based protocol for the isolation of ECs and the Eng gene deletion strategy. ( B ) Confocal images of cultured ECs isolated from lungs and liver and stained for PECAM1, VE-Cadherin and Endoglin (Scale bar: 10 μm). ( C ) Confocal images of lung ECs stained for endoglin at different time points after Eng gene deletion and fluorescence intensity quantification of its expression at the endothelial cell surface (a minimum of 10 cells were quantified per time point) (Scale bar: 10 μm). ( D ) Confocal images of lung and liver ECs stained for PECAM1, VE-Cadherin and Endoglin at 0, 20 and 60 h after viral infection, showing the efficacy of the Eng gene deletion (Scale bar: 10 μm) and fluorescence intensity quantification of endoglin expression at the endothelial cell surface (a minimum of 35 cells were quantified per time point). ( E ) ECs isolated from lungs and liver of P7 pups were exposed to VEGF (25 ng.ml −1 ), TGF-β1 (1 ng.ml −1 ) and BMP9 (1 ng.ml −1 ) for 30 min at 37 °C before lysis. Whole cell extracts were fractionated by SDS-page and blotted. The filters were incubated with Phospho-Akt, Phospho-Smad1, Akt, Smad1, endoglin and β-actin. Representative results from at least 4 independent experiments are shown. Graphs represent quantification of the Western blotting. Error bars represent SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 result from 1-way ANOVA and Dunnett’s post hoc tests comparing the mean of each group to the unstimulated condition. ns: Not significant. ( F ) Western blot analysis of Akt, p42/p44 MAPK and Smad1 phosphorylation at different time points after Eng gene deletion.

Article Snippet: The ear microvasculature was labelled with rat anti-mouse Pecam1 (Clone 13.3) (550274, BD Pharmigen, Franklin Lake, NJ, USA) or with rat anti-mouse endoglin (clone 2Q1707) (sc-71042, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and then incubated with Alexa Fluor ® 555 donkey anti-rat (A-21434, Invitrogen, Waltham, MA, USA) and with monoclonal anti-α-smooth muscle actin conjugated with FITC (clone 1A4) (F3777, Sigma-Aldrich, Saint louis, MO, USA).

Techniques: Protein-Protein interactions, Isolation, Cell Culture, Staining, Fluorescence, Expressing, Infection, Lysis, SDS Page, Incubation, Western Blot, Phospho-proteomics

Journal: International Journal of Molecular Sciences

Article Title: Thresholds of Endoglin Expression in Endothelial Cells Explains Vascular Etiology in Hereditary Hemorrhagic Telangiectasia Type 1

doi: 10.3390/ijms22168948

Figure Lengend Snippet: Schematic representation of how heterogeneity in endoglin levels in ECs predict the tissue-specific manifestations of HHT1.

Article Snippet: The ear microvasculature was labelled with rat anti-mouse Pecam1 (Clone 13.3) (550274, BD Pharmigen, Franklin Lake, NJ, USA) or with rat anti-mouse endoglin (clone 2Q1707) (sc-71042, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and then incubated with Alexa Fluor ® 555 donkey anti-rat (A-21434, Invitrogen, Waltham, MA, USA) and with monoclonal anti-α-smooth muscle actin conjugated with FITC (clone 1A4) (F3777, Sigma-Aldrich, Saint louis, MO, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Thresholds of Endoglin Expression in Endothelial Cells Explains Vascular Etiology in Hereditary Hemorrhagic Telangiectasia Type 1

doi: 10.3390/ijms22168948

Figure Lengend Snippet: Primers and conditions.

Article Snippet: The ear microvasculature was labelled with rat anti-mouse Pecam1 (Clone 13.3) (550274, BD Pharmigen, Franklin Lake, NJ, USA) or with rat anti-mouse endoglin (clone 2Q1707) (sc-71042, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and then incubated with Alexa Fluor ® 555 donkey anti-rat (A-21434, Invitrogen, Waltham, MA, USA) and with monoclonal anti-α-smooth muscle actin conjugated with FITC (clone 1A4) (F3777, Sigma-Aldrich, Saint louis, MO, USA).

Techniques: Sequencing