Journal: bioRxiv

Article Title: Targeting NRP1 with EG00229 induces neurovascular permeability

doi: 10.1101/2023.11.07.564946

Figure Lengend Snippet: Freshly dissected retinae from mice of the indicated genotypes were incubated in Krebs solution with or without VEGF164 or EG00229, fixed and immunostained with the vascular EC marker isolectin B4 (green) and an antibody against P-p38 (magenta). tinas were obtained from the second eye of mice used for ex vivo permeability experiments in . In ( A-C ), we used retinas from adult Kdr fl/fl ( A ), Flt1 fl/fl ( B ) and Nrp1 fl/fl ( C ) mice expressing or lacking the endothelial Cdh5 -CreER T2 transgene, which had been tamoxifen-treated two weeks before ex vivo treatment of the retina. In ( D ), we used retinas from Nrp1 D320K/D320K and wild type littermate mice for ex vivo treatment. Re Epifluorescent images (scale bars: 10 μm) show P-p38 in IB4-positive vasculature and IB4-negative neuroglial cells. Epifluorescent images were then used for quantification of P-p38 pixel intensity in the IB4-positive area. Data are shown as mean ± SD; each data point represents the value for one retina from one mouse; n = 3 mice for each genotype. Significant P-values are shown for P-p38 staining area for VEGF164 or EG00229 versus vehicle (Krebs) for each genotype (asterisks) and for VEGF164 or EG00229 in mutant versus control (hashtags) (two-way ANOVA): *, P < 0.05; **, P < 0.01; ***, P < 0.001; #, P < 0.05; ns, not significant (P > 0.05).

Article Snippet: Mouse retinae were fixed in 4% formaldehyde in PBS for 1h and wholemount immunostained as described [ ] using rabbit anti-P-p38 MAPK (Thr180/Tyr182) antibody (Cell Signaling) followed by Alexa Fluor 555–conjugated goat anti–rabbit antibodies (Invitrogen) and biotin-conjugated IB4 (Merck) followed by Alexa Fluor 488-conjugated streptavidin (Invitrogen).

Techniques: Incubation, Marker, Ex Vivo, Permeability, Expressing, Staining, Mutagenesis

Journal: bioRxiv

Article Title: Targeting NRP1 with EG00229 induces neurovascular permeability

doi: 10.1101/2023.11.07.564946

Figure Lengend Snippet: (A) Quantification of sulforhodamine B fluorescence changes in the ex vivo retina permeability assay after treatment with SU1498, SB203580 or PP2 (inhibitors) alone, compared to VEGF164 or EG00229 treatement with or without inhibitor pre-treatment (n = 3 treatments of 3 independent retinas each). Data are shown as mean ± SD; each data point represents the value for one retina from one mouse. Significant P-values are shown for inhibitor versus baseline (asterisks) and EG00229 ± inhibitor (hashtags): *** P, < 0.001; ###, P < 0.001; ns, not significant (P > 0.05). Repeated measures two-way ANOVA. ( B,C ) Freshly dissected retinae from wild type mice were incubated in Krebs solution (vehicle) with or without EG00229, or preincubated with SB203580 or PP2 for 15 min before adding EG00229. Ex vivo retinae were then fixed and immunostained with the vascular EC marker isolectin B4 (IB4, green) and an antibody against P-p38 (magenta). Epifluorescent images ( B , scale bars: 10 μm) were used for quantification of pixel intensity for P-p38 in the IB4-positive vascular area ( C ). Significant P-values for P-p38 staining in the IB4+ area for inhibitors (SB203580 or PP2 versus SB203580) versus EG00229 are indicated with asterisks or after pre-treatment with the inhibitors, indicated with hashtags: *, P < 0.05; ***, P < 0.001; ###, P < 0.001; ns, not significant (P > 0.05). One-way ANOVA.

Article Snippet: Mouse retinae were fixed in 4% formaldehyde in PBS for 1h and wholemount immunostained as described [ ] using rabbit anti-P-p38 MAPK (Thr180/Tyr182) antibody (Cell Signaling) followed by Alexa Fluor 555–conjugated goat anti–rabbit antibodies (Invitrogen) and biotin-conjugated IB4 (Merck) followed by Alexa Fluor 488-conjugated streptavidin (Invitrogen).

Techniques: Fluorescence, Ex Vivo, Permeability, Incubation, Marker, Staining