Dual-Probe In-Situ Hybridization Protocol

The digoxin-labeled probes specific to circ_001680 and biotin-labeled probes against miR-340 were prepared by Geneseed Biotech, and the sequences are showen in Additional file 1 Table S1. SW480 and HCT116 cells were cultured on coverslips and fixed with 4% paraformaldehyde in PBS for 15 min. The probes were diluted in hybridization solution (Geneseed Biotech, Guangzhou, China) in PCR tubes and were heated at 95 °C for 2 min in a PCR block to denature the probe. The probe was immediately chilled on ice to prevent reannealing. The hybridization solution was drained, and 100 μL of diluted probe per section was added to cover the entire sample. The samples were covered with a coverslip to prevent evaporation and were incubated in the humidified hybridization chamber at 65 °C overnight. The signals were detected by Cy3-conjugated anti-digoxin and FITC-conjugated anti-biotin antibodies (Jackson ImmunoResearch Inc., West Grove, PA, USA). Cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Finally, the images were obtained on a Zeiss LSM 700 confocal microscope (Carl Zeiss, Oberkochen, Germany) [23 (link)–26 ].