5 bromo 4 chloro 3 indolylphosphate nitroblue tetrazolium bcip nbt

For ISH, a specific probe targeting the splicing site of circEHD2 was labeled with 5′-digoxin (DIG) and 3′- digoxin (DIG), and the probe was designed and synthesized by Servicebio (Wuhan, China). The scramble probe and U6 probe were used as a negative control and internal control, respectively. Briefly, the paraffin sections were deparaffinized and dehydrated by xylene and a series of graded ethanol, followed by incubation with proteinase K at 37 °C for 20 min and with Triton-X100 at 4 °C for 10 min. The sections were incubated with hybridization buffer containing the circEHD2 probe at 37 °C overnight, followed by incubation with the anti-digoxin antibody at 4 °C overnight. Then, the sections were stained with 5-Bromo-4-Chloro-3-Indolylphosphate/Nitroblue Tetrazolium (BCIP/NBT) (Beyotime, Shanghai, China) for 30 min at room temperature, followed by staining with Nuclear fast red (Servicebio, Wuhan, China) for 3 min at room temperature. The images were photographed by an Olympus microscope (Olympus, Tokyo, Japan). The H-score of circEHD2 was assessed as follows: H-score = Σ (P × I), where P represents the percentage of stained cells, and I indicates the staining intensity score as follows: 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (intense staining). The probes used for ISH are listed in the Table S4.