Dp2 bsw image acquisition system

Tg (vegfr2:GFP) zebrafish embryos 24 h post-fertilization were treated with 1 mg/mL pronase E solution to remove the egg membrane. Then, they were randomly divided into 4 groups, namely the normal control group, the model group (vatalanib, PTK787), the positive control group (Danhong injection), and the experimental group. Each group had 10 zebrafish embryos, and each group had two parallel repeats.
As shown in Figure 8A, the model group was built successfully based on significant inhibition of the growth of intersegmental blood vessels (ISVs) by treating zebrafish embryos with vatalanib (PTK787). Then, the positive control group and experimental group were also treated with vatalanib PTK787 (0.2 μg/mL) to afford model zebrafish whose intersegmental blood vessels (ISVs) were significantly inhibited. Simultaneously, the positive control group was given a Danhong injection (10 μL/mL) and the experimental group was given various concentrations of tested compounds (20 μM, 40 μM, 80 μM).
After incubation at 28 °C for 24 h, the intersegmental blood vessels (ISVs) of zebrafish were observed and visualized under a fluorescence microscope (SZX16 stereo microscope and DP2-BSW image acquisition system, Olympus, Japan), and the ISV index was calculated as follows: ISV index = number of intact vessels × 1 + number of defective vessels × 0.5 [12 (link)].

Healthy and limpid embryos were picked out at 24 h post-fertilization (hpf) and distributed into a 24-well microplate (6–8 embryos/well) in 1 mL Holtfreter’s solution and maintained at 28 °C. The sample solution was diluted with embryo water to different concentrations of 0.5, 1, 10, 100 μg/mL, and added into the well. The final volume of each well was 2.0 mL, and the content of DMSO in each well was adjusted to be consistent. 2.0 mL 0.1 μg/mL PTK787 solution served as positive controls. The embryo water or DMSO (0.5%, V/V) served as blank controls. Embryos were maintained in incubator at 28 °C for additional 48 h, placed onto a glass slide, photographed using SZX16 fluorescence stereomicroscope and DP2-BSW image acquisition system (Olympus, Japan) after anesthesia. Zebrafish somite intersegmental vessels (ISVs) were quantified using Image Pro Plus software. Anti-angiogenic effects were defined as decrease of SIVs length [14 (link)].

Six samples from SCV in each group were deparaffinized in xylene and rehydrated by a series of ethanol concentrations from 100% to 75%. Sections were subsequently immersed in 3% hydrogen peroxide solution to inhibit endogenous peroxidase activities and incubated in 10% normal goat serum. Finally, sections were incubated at 4°C overnight against the following primary antibodies: anti-Cadherin 5 (1:100, Boster Biological Technology, China), anti-CD34 (1:200, Abcam, UK), anti-VEGF (1:200, Abcam), anti-CTSD (1:200, Proteintech, USA), anti-SOD1 (1:200, Proteintech), and anti-C-CASP3 [1:200, CST (Cell Signaling Technologies, USA)]. These sections were then incubated with HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, USA) and counterstained with hematoxylin. Each specimen was imaged at a magnification of ×200 using a DP2-BSW image-acquisition system (Olympus Corp), and absorption values quantified using the Image-Pro Plus software (Media Cybernetics, MD, USA) to assess expressions of Cadherin 5, VEGF, CTSD, SOD1, and C-CASP3 and the number of CD34 positive microvessels.