2-Aminoethyl diphenylborinate  别名：2-APB; diphenylboric acid-2-aminoethyl ester; DPBA

2-Aminoethyl diphenylborinate（2-APB）是一种具有细胞通透性的IP3R抑制剂，可以调节IP3诱导的钙释放。2-Aminoethyl diphenylborinate（2-APB）还可以抑制SOC通道的活性，并且在高浓度的时候可以激活TRP通道。2-Aminoethyl diphenylborinate (100 μM，9 小时) 对缺血再灌注 (I/R) 引起的 Hep G2 细胞损伤具有保护作用。

2-Aminoethyl diphenylborinate (2 或 4 mg/kg，腹腔注射，单次给药 10 分钟) 在缺血后再灌注诱导的睾丸损伤大鼠模型中减少了细胞的氧化应激和凋亡。2-Aminoethyl diphenylborinate (2 mg/kg，经门静脉注射，缺血处理前 10 分钟注射) 在缺血后再灌注引起肝脏损伤的大鼠模型中，可以有效预防肝脏 I/R 损伤。

DPBA staining solution preparation

Reagent Amount for 100 ml
2-Aminoethyl diphenylborinate (DPBA) 0.25 g
Triton X-100 20 μl
Milli-Q water  up to 100 ml
Notes:
a. Some crystals of DPBA can be retained and seen after mixing.
b. This staining solution can be stored at -20 °C for further uses.

Equipment

1. Tweezers
2. A plant growth chamber
3. Micro-tubes rotator
4. Pipette
5. Confocal microscopy system

Plant growth and DPBA staining procedure

1. Here, Arabidopsis thaliana is used. However, this protocol can be applied for other plants as well. Grow plants on MS medium supplemented with 2% sucrose and 1.2% phyto-agar for 5 days. The growth chamber conditions were 22 ± 1 °C, long-day (16 h light/8 h dark), and light intensity 100 μmol m^-2 s^-1.
Note: After seeding, remember to place the medium plates vertically in the growth chamber. Thereby, the roots grow on the surface of the medium and intact root tip samples can be obtained.
2. Add 1 ml of DPBA staining solution (Recipe 1) to each micro-tube.
3. Use a clean tweezers to carefully transfer the 5-day-old Arabidopsis seedlings to the micro-tube containing DPBA staining solution (5 seedlings/micro-tube).
4. Place the micro-tube in the rotator and rotate for 5 min at room temperature.
5. Stop the rotator, transfer the micro-tube to a rack and remove the DPBA staining solution.
Note: At this step, carefully use a pipette to remove the DPBA staining solution and try to not damage the plants, especially the root tips.
6. For washing, add 1.5 ml of distilled water to each micro-tube. Place the micro-tube in the rotator and rotate for 2 min at room temperature.
7. Stop the rotator, transfer the micro-tube to a rack and remove water.
8. Repeat Steps A6 and A7 two more times.
Note: After washing, can keep the plants in water and try to detect immediately. Do not leave the samples staying in water for longer than 30 min.
9. Transfer the seedlings to a microscope slide, cover it and detect the flavonols accumulation in the root tips by a confocal microscope.

Confocal microscopy

1. For DPBA-kaempferol, apply the emission spectrum (475-500 nm).
2. For DPBA-quercetin, apply the emission spectrum (585-619 nm).
3. Remember to take an additional bright field picture for control.