Duolink in situ detection reagent green

Cells grown on coverslips were fixed by addition of 10% formaldehyde to the final concentration of 2%, permeabilized in 0.1% Triton X-100 for 10 min and blocked with 3% (w/v) BSA/PBS at 4°C overnight. Cells were labeled with primary and corresponding Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibody (Invitrogen) for 1 h at room temperature. Cells were then mounted with Aqua-Poly-Mount mounting medium (Polysciences) and analyzed on a Leica TCS-SP2 AOBS confocal microscope. For immunostaining of mice Aortic VSMC, cells were incubated with 5% mouse serum in PBS followed by 1 h incubation with 5% normal goat serum.
Duolink In Situ Proximity Ligation Assay (PLA) Probes (anti-rabbit PLA probe PLUS, anti-mouse PLA probe MINUS, and Duolink In Situ Detection Reagent Green) were purchased from Sigma-Aldrich and used accordingly to the manufacturer’s instructions. The number of PLA signals was quantified using ImageJ software.

To examine SL-13R-protein interaction, proximity link assay was performed. UCB CD34+ cells were cultured with biotin-conjugated SL-13R for 48 h. Cytospin, cell fixation and permeabilization are similar to previous mention in Immunocytochemistry. After overnight incubation with rabbit anti-AHNAK, ANXA2, and PLEC antibody (Table S4) and mouse anti-biotin antibody, Duolink In Situ PLA Probe anti-rabbit MINUS and anti-mouse PLUS were applied to samples (Sigma-Aldrich, Uppsala, Sweden). Proximally located antibody-binding probes were ligated with oligonucleotide by ligase at 37℃ for 30 min using Duolink In Situ Detection Reagent Green (Sigma-Aldrich, Uppsala, Sweden). Rolling Circle Amplification of probes was performed using DNA polymerase at 37℃ for 100 min. After wash with Buffer B, nuclei were stained with TOTO-3, and analysed using a FluoView 1000 Confocal Microscope.

Proximity ligation assay was performed in a humid chamber at 37 °C according to the manufacturer’s instructions (Duolink In Situ detection reagent-Green, Sigma Aldrich). Cells were seeded on glass coverslips and allowed to attach and transferred to hypoxia. They were then fixed with 4% paraformaldehyde and permeabilized with 0.2% Tween-20 in PBS. Paraffin-embedded CRC tissues (Ethics approval EK/BmV-02/2016) were deparaffinized, rehydrated and antigen retrieval was performed using citrate unmasking solution. Samples were blocked with 3% BSA/PBS for 30 min, incubated with a mixture of antibodies against CAIX (NB100-417, Novus Biological, Centennial, Colorado; diluted to 1 µg/mL) and PIMT (sc-100977, Santa Cruz Biotechnology, Dallas, TX, USA; diluted to 2 µg/mL) or CAIX (M75, diluted to 1 µg/mL) and AE2 (GenScript, Piscataway, NJ, USA; diluted to 1 µg/mL) for 1 h, incubated with plus and minus PLA probes for 1 h, then incubated with a ligation mixture containing connector oligonucleotides for 30 min, and finally with an amplification mixture containing a fluorescently labelled DNA probe for 100 min. The samples were analyzed by laser scanning confocal microscopy (LSM 510 Meta Microscope; Zeiss, Oberkochen, Germany). All PLA stainings were performed in two independent experiments and representative images of each analysis are shown in figures.