Scientists identify how some angiogenic drugs used to treat cancer and heart disease cause vascular disease
. Mouse IgG was used in littermates as a control treatment. For the 48-h experiment, mice received a single intraperitoneal injection of 200 µl of IgG or anti-Dll4 antibody . For the 2-week blocking experiments, mice received anti-Dll4 antibody or anti-NRR1 antibody four times over 14 days at a concentration of 7.5 mg kg, respectively.
We used mice with C57BL/6 or C57BL/6×129SV genetic backgrounds. To generate mice for analysis, we intercrossed mice with an age range of 7–30 weeks. Mice used for experiments were 2–5 months old. We do not expect our data to be influenced by mouse sex.mice, we used guide RNAs Notch4_1 and Notch4_2 to target mouse Notch4 in mouse eggs from the C57BL/6 genetic background. Injection mixture was composed by the described CRISPR RNA and trans-activating CRISPR RNA at 0.
Tissues were fixed for 2 h in 4% PFA in PBS at 4 °C. After three washes in PBS for 10 min each, organs were stored overnight in 30% sucrose in PBS. Organs were then embedded in OCT and frozen at −80 °C. Cryosections were cut on a cryostat , washed three times for 10 min each in PBS, and blocked and permeabilized in PBS containing 10% donkey serum , 10% fetal bovine serum and 1% Triton X-100.
. To detect Ki67 or c-Myc in the same section as ERG, we used rabbit anti-Ki67 or anti-c-Myc together with a Fab fragment Cy3 secondary antibody, which is compatible with the later use of rabbit anti-ERG conjugated to Alexa Fluor 647.Tissues were fixed for 2 h in 4% PFA in PBS and washed as above. Organs were then embedded in 6% agarose low-melting gel , and organ sections were cut on a vibratome. Sections were permeabilized for 1 h in PBS containing 1% Triton X-100 and 0.5% Tween 20.
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