Dapi containing fluorescent mounting media

Sub-confluent cells cultured on glass coverslips in 24-well plates were washed with PBS and fixed with 4% paraformaldehyde for 15 min. After two washes with PBS, the cells were incubated with a blocking buffer (2% BSA in PBS) before incubation with mouse anti-galectin-3 monoclonal antibody and rabbit anti-MCAM polyclonal antibody (Proteintech) (1:500 and 1:1000 dilution, respectively, in 1% BSA in PBS) for 2 h at room temperature. Control wells were incubated with 1% BSA in PBS. After three washes with PBS, the cells were incubated with Texas Red-conjugated anti-mouse antibody (Vector Laboratories, Burlingame, CA, USA) at 1:300 dilution and FITC-conjugated anti-rabbit (Dako Agilent, Santa Clara, CA, USA) at 1:300 dilution for 1 h at room temperature. The cells were washed five times with PBS before being mounted with DAPI-containing fluorescent mounting media (Vector Laboratories, Burlingame, CA, USA) and analysed by a Zeiss LSM 800 Airyscan confocal microscope.
In some experiments, the cells were treated with 10 µg/mL recombinant galectin-3 or 10 µg/mL BSA for 1 h before they were fixed and analysed for MCAM and galectin-3 localization by confocal microscopy.

Sub-confluent cells grown on glass coverslips in 24-well plates were incubated in serum-free at 37 °C overnight. The cells were treated with BSA (2 μg/ml) (control), EGF (20 ng/ml) without or with galectin-3 (2 μg/ml) for 10 min at 37 ^°C. The cells were washed with ice-cold PBS and fixed with 4% paraformaldehyde. The cells were then washed again with PBS and probed with anti-MUC1 B27.29 (1 μg/ml) or anti-EGFR (D38B1) (2 μg/ml) for 2 h at room temperature. After two washes with PBS, FITC-conjugated anti-mouse or Alexa fluor 643-conjugated anti-rabbit antibodies were applied for 1 h at room temperature. The cells were washed twice before being mounted using DAPI-containing fluorescent mounting media (Vector Laboratories, Burlingame, CA, USA). The slides were analysed using a 3i confocal microscope (Marianas SDC, 3i Imaging) and Slidebook 6 Reader version 6.0.4 (Intelligent-imaging).

Sub-confluent HUVECs cultured on glass coverslips in 12-well plates were fixed with 4% paraformaldehyde. After incubation with 2% BSA/PBS for 1 h, the cells were incubated with 4 μg/ml FITC-PNA, antibodies to MCAM or PECAM (1:1000) in 1% BSA/PBS for 1 h at room temperature. After three washes with PBS, the cells were incubated with Alexa fluor 643-conjugated secondary antibody for 1 h at room temperature. The cells were washed five times with PBS before being mounted with DAPI-containing fluorescent mounting media (Vector Laboratories, Burlingame, CA) and analysed by confocal microscopy (Marianas SDC, 3i Imaging).