Ab10482

Cells were washed with 1x PBS and fixed for 20 min in paraformaldehyde (PFA) 4% at room temperature. After two washes with 1x PBS, they were permeabilized for 20 min in 1x PBS - 0.025% saponin and then blocked for 30 min in 1x PBS - 0.025% Saponin - 1% BSA (Bovine Serum Albumin, Sigma-Aldrich). Cells are incubated overnight at 4 °C in 1x PBS - 0.025% saponin, 1% BSA containing the primary antibody. The following day, after washing with 1x PBS, cells were incubated in 1x PBS - 0.025% saponin - 1% BSA containing the secondary antibody coupled to a fluorochrome for 1 h protected from light at room temperature. After two washes with 1x PBS, the nuclei were stained with 10 μg.ml⁻¹ Hoechst33342 (H357C, Invitrogen) for 20 min. Slides were washed with 1x PBS, then with distilled water before being mounted on slides with Fluoromouont-G (0100-01, SouthernBiotech). The slides were visualized using an SP5 confocal scanning Tandem RS (Leica) and analyzed by ImageJ. The following antibodies were used: anti-γ-tubulin (T6557, Sigma-Aldrich); anti-GM130 (610823, BD Biosciences); anti-E-cadherin (ab1416, Abcam); anti-E-cadherin (610181, BD Biosciences); anti-Galectin-7 (ab10482, Abcam); anti-β-catenin (MA1-301, Thermo Scientific); anti-α-catenin (13-9700, Thermo Scientific); anti-α-catenin (ab51032, Abcam); anti-S100A11 (ab180593, Abcam).

Tissue processing and immunostaining were performed as previously described^{32 (link)}. The following primary antibodies were used: rabbit polyclonal anti-galectin-7 antibodies (ab10482, Abcam) and mouse monoclonal anti-β-catenin antibody (MA1-301, Thermo Scientific).
Nuclei were stained with Hoechst33342 (H3570, Invitrogen) and confocal acquisition was performed using a Leica SP5 microscope. Pearson’s r coefficient was measured using JACoP plugin from ImageJ on a selection including all the layers of the epidermis.