Confocal fluorescence microscopy

The EVs derived from RCC cells were labeled by using the PKH26 Red Fluorescent Cell Linker Kit (Umibio, Shanghai, China). Then MRC5 cells were incubated with the PKH26-labeled EVs in the dark overnight. After the cells were stained by 4′,6-diamidino-2-phenylindole (DAPI), confocal fluorescence microscopy (Carl Zeiss AG, Jenna, Germany) was used to observe the internalization of EVs.
For the internalization of circEHD2-ASO, Cy3-labeled circEHD2-ASO was incubated with human RCC cells (OSRC-2 and 786-O) in the dark for 24 h. Then, the PKH67 Green Fluorescent cytomembrane Linker Kit (Solarbio, Beijing, China) was used to label the cytomembrane of RCC cells at 4℃ for 20 min. Next, the cells were washed twice with phosphate buffered saline (PBS), before DAPI was used to stain the nuclei of the RCC cells. The internalization of circEHD2-ASO in RCC cells was photographed by confocal fluorescence microscopy (Carl Zeiss AG, Jenna, Germany). The sequence of circEHD2-ASO was described in the Table S2.

For immunofluorescence, tissues were permeabilized in 0.1% PBS-T for 15 min. To retrieve antigens, the samples were boiled in 0.1 M citrate buffered saline (pH 6.0) for 5 min. The sections were cooled for 30 min and then washed twice in PBS (5 min each). After washing in PBS-T for 30 min, the sections were blocked for 1 h in blocking solution (4% normal donkey serum in PBS-T). The sections were incubated with primary antibody overnight at 4 °C. Anti-oligomer A11 (Invitrogen, CA, USA) (1:100), Anti-6E10 (Aβ_1–16) (Covance, NJ, USA) (1:500), Anti- D54D2 (Aβ_1–40, Aβ_1–42) (Cell Signaling, MA, USA) (1:500), anti-synaptophysin (Agilent Dako, CA, USA) (1:250), anti-TH (Santa Cruz Biotech, TX, USA) (1:250), and anti-Ki67 (Cell Signaling, MA, USA) (1:250) antibodies were used. Alexa 488 and Cy3-conjugated secondary antibodies (Jackson Laboratory, Bar Harbor, ME, USA) were used. The sections were counter-stained and mounted using VECTASHIELD mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, CA, USA). The images were visualized and photographed by confocal fluorescence microscopy (Carl Zeiss, Thornwood, NY, USA).