To induce endorepellin overexpression in the endothelium, tamoxifen (2 mg dissolved in corn oil; Sigma, H7904) was administered i.p. for 5 consecutive days in Tie2CreERT2;ERKi mice. After 3 days, mouse thoracic aortae were dissected and cross-sectioned into 1–2 mm rings. Rings were sandwiched between Collagen type I (1 mg/ml) and cultured in endothelial cell media at 37°C for 5–7 days as described previously [83 ,84 ,114 ]. Following fixation with 4% (w/v) paraformaldehyde and blocking in 2% (w/v) BSA (45 min), rings were incubated overnight at 4°C with anti-perlecan domain V antibody (1:50, custom rabbit antibody) then incubated with Alexa Fluor 594 donkey anti-rabbit H+L IgG (R37119, 1:400; ThermoFisher) and Alexa Fluor 488 Isolectin GS-IB4 (I21411, 1:200; ThermoFisher) for 3 h at RT. Confocal analysis via Zeiss LSM780 NLO multiphoton microscope and quantification of fluorescence intensity of angiogenic sprouts peripheral to the ring were performed as previously described [67 (link),83 ,84 ].
Alexa fluor 488 isolectin gs ib4
Manufactured by Thermo Fisher Scientific
Sourced in China
Alexa Fluor 488 Isolectin GS-IB4 is a fluorescent conjugate of a lectin derived from the seeds of the Griffonia simplicifolia plant. It binds specifically to α-D-galactose and N-acetyl-α-D-galactosamine residues, and is commonly used to label endothelial cells and vasculature in biological samples.
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2 protocols using alexa fluor 488 isolectin gs ib4
1
Induction of Endorepellin Overexpression in Endothelium
2
Retinal Angiogenesis Inhibition Assay
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Postnatal day 4 (P4) pups were i.p. injected with 15–20 µL of phosphate buffered saline (PBS), anti-BMP9 (BMP9 blocking antibody, 200 mg/mL, sc-27821, Santa Cruz Biotechnologies, Shanghai, China), and NIBR0213 (30 mg/kg), respectively. Then eyeballs from P7 pups were isolated and fixed with 4% paraformaldehyde (PFA) 4 h at 4 °C and surgical dissection of retina was performed under stereoscopic microscope. The isolated retinas were stained with Alexa Fluor® 488 isolectin GS-IB4 (Thermo Fisher Scientific, Shanghai, China) mounted on a microscope slide in the presence of an anti-fluorescence quenching agent. The retinal blood vessels were observed and photographed using the EVOS FL Auto imaging system (Thermo Fisher Scientific, Shanghai, China). Angiotool 0.5a software (https://ccrod.cancer.gov/confluence/display/ROB2/Home ) was employed to analyze angiogenesis-related parameters, include vessels percentage area (the percentage of area occupied by vessels inside the explant area), junctions density (the number of vessel junctions normalized per unit area), and average lacunarity (mean lacunarity overall size boxes) (25 (link)).
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