Duolink reagent kit

The interaction between Gal-3 and CEA was determined by the Duolink reagent kit (Olink Biosciences, Uppsala, Sweden). A total of 1x10³ cells were seeded onto a chamber slide. Cells were fixed in 1% paraformaldehyde and washed with wash buffer A. Cells were then incubated with blocking solution at 37°C for 30 min followed by incubation with primary antibodies, mouse anti Galectin-3 (Santa Cruz) and rabbit anti CEA (Santa Cruz) at 37°C for 60 min. The cells were further washed and incubated with th corresponding PLA probe (anti mouse PLUS and anti rabbit MINUS) at 37°C for 60 min. The probes were then ligated with ligation solution and amplified with amplification solution. The slides were imaged by fluorescence microscopy (Axio Observer Z1, Carl Zeiss MicroImaging, Inc., Welwyn Garden City, UK).

We used a Duolink^® reagent kit (Olink Biosciences) to investigate in situ protein-protein interactions. The procedures for the administration of primary antibodies (p53 versus MDM2 or p53 versus 14-3-3σ), PLA probes, hybridization, ligation, amplification, detection, and mounting were performed according to the manufacturer's protocol. The images were captured using a fluorescence microscope (Axio Observer Z1, Carl Zeiss MicroImaging, Inc., Welwyn Garden City, UK).