Journal: Genes to Cells

Article Title: Attenuation of Oxygen‐Induced Neovascularization and Inflammation by Neutralizing VEGFA and/or ANG ‐2 With an Antibody

doi: 10.1111/gtc.70107

Figure Lengend Snippet: Effects of anti‐VEGFA, ‐ANG‐2, or bispecific antibody on pathological neovascularization in the mouse OIR model. (A) Schematic representation of the experimental design to evaluate the effects of intraocular injected antibodies in a mouse OIR model. The antibodies were intravitreally injected into OIR mice at P14. Retinal whole‐mounts were prepared at P17 and P19 and immunostained with anti‐CD31 antibody. (B) The areas of neovascularization (red) and vaso‐obliteration (non‐perfusion, yellow) in the OIR retina treated with control IgG, anti‐VEGFA antibody, anti‐ANG‐2 antibody, or anti‐VEGFA/ANG‐2 bispecific antibody are shown. Scale bar = 1 mm. (C and D) The area (% of total area) of neovascularization and the vaso‐obliteration area in the OIR mouse retina. The averages of the area with standard errors are shown in the bar graphs. (E and F) The antibodies were intravitreally injected into OIR mice at P14, and the retina was harvested at P17 and frozen sectioned. Immunostaining with anti‐CD31 and ‐Ki67 antibodies was performed to examine endothelial cell proliferation. Scale bar = 50 μm. Nuclei were visualized with DAPI staining. Numbers of CD31 and Ki67 double‐positive cells inner to the INL were counted (F). Data are the averages of 8 samples with standard deviation. (C, D, F) Tukey's HSD test was used for the statistical analysis. * p < 0.05; ** p < 0.01.

Article Snippet: Recombinant VEGFA (Thermo Fisher Scientific, 450‐32) and ANG‐2 (R&D SYSTEMS, 7186‐AN‐025) were labeled with Alexa Fluor 488 using a Microscale Protein Labeling Kit (Thermo Fisher Scientific, A30006).

Techniques: Injection, Control, Immunostaining, Staining, Standard Deviation

Journal: Genes to Cells

Article Title: Attenuation of Oxygen‐Induced Neovascularization and Inflammation by Neutralizing VEGFA and/or ANG ‐2 With an Antibody

doi: 10.1111/gtc.70107

Figure Lengend Snippet: Gene expression patterns of the OIR retina administered antibodies at P17 and P19. RNAseq was performed using whole RNA purified from the retinas of normal mice and OIR mice administered the antibodies. Four samples of each condition were prepared. (A) Volcano plots are shown. Upregulated genes (≥ log2 (1.5)) are indicated as red circles, and downregulated genes (≤ − log2 (1.5)) are indicated as blue circles. (B–E) TPM values of specific genes are shown. A2, ANG‐2 antibody; C, control; V, VEGFA antibody; VA2, Bispecific antibody. Tukey's HSD test was used for the statistical analysis. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Recombinant VEGFA (Thermo Fisher Scientific, 450‐32) and ANG‐2 (R&D SYSTEMS, 7186‐AN‐025) were labeled with Alexa Fluor 488 using a Microscale Protein Labeling Kit (Thermo Fisher Scientific, A30006).

Techniques: Gene Expression, RNA sequencing, Purification, Control

Journal: Genes to Cells

Article Title: Attenuation of Oxygen‐Induced Neovascularization and Inflammation by Neutralizing VEGFA and/or ANG ‐2 With an Antibody

doi: 10.1111/gtc.70107

Figure Lengend Snippet: Changes of IBA1‐positive cells and the effects of intraocular injected anti‐VEGFA, ‐ANG‐2, or bispecific antibody. (A) Retinal whole‐mounts of the OIR mice at P17 and P19 treated with control IgG, anti‐VEGFA, ‐ANG‐2, or bispecific antibody were immunostained with anti‐IBA1 antibody and observed by a confocal microscope. Scale bar = 1 mm. (B) The IBA1 signal‐positive area was examined using ImageJ software, and the percentage of the positive area to the whole area is shown in (B). The averages of 3 independent samples with standard deviation are shown. * p < 0.05; ** p < 0.01. (C) Alexa Fluor (AF) 488‐conjugated VEGFA or ANG‐2 was intravitreally injected into OIR mice at P17. The eyes were harvested one hour after the injection, and whole‐mounts were immunostained with anti‐CD31 and anti‐IBA1 antibodies and observed by a confocal microscope. White arrowheads indicate VEGFA‐ or ANG‐2‐Alexa Fluor 488 signals in IBA1‐positive cells. Scale bar = 20 μm.

Article Snippet: Recombinant VEGFA (Thermo Fisher Scientific, 450‐32) and ANG‐2 (R&D SYSTEMS, 7186‐AN‐025) were labeled with Alexa Fluor 488 using a Microscale Protein Labeling Kit (Thermo Fisher Scientific, A30006).

Techniques: Injection, Control, Microscopy, Software, Standard Deviation