Immunofluorescence Staining of 3D Cells

S1, miR-183-S1, miR-492-S1 and miR-Control-S1cells were plated on 4-well chamber slides (3D) and were stained by immunofluorescence on day 11 as described by Fostok et al.^{30 (link)}. Briefly, cells were washed with 1 × PBS and permeabilized with 0.5% peroxide and carbonyl-free Triton X-100 in cytoskeleton buffer (100 mM NaCl, 300 mM sucrose, 10 mM PIPES, pH 6.8, 5 mM MgCl2, 1 mM pefabloc, 10 μg/mL aprotinin, 250 μM NaF). Cells were washed twice with cytoskeleton buffer and fixed in 4% formaldehyde. Cells were subsequently washed three times with 50 mM glycine in 1 × PBS and blocked. Primary antibodies used were mouse monoclonal β-catenin (200 μg/mL; Santa Cruz Biotechnology, Dallas, TX, USA, sc-7963), mouse monoclonal Scrib (200 μg/mL; Santa Cruz Biotechnology, Dallas, TX, USA, sc-55543) and mouse monoclonal Connexin 43 (200 μg/mL; Santa Cruz Biotechnology, Dallas, TX, USA, sc-271837) all at dilutions (1:200). Secondary antibody conjugated to Alexa Fluor 568 (red), goat anti-mouse (Invitrogen, Waltham, MA, USA, A-11004) was used according to the manufacturer's recommended dilutions (1:1000). Nuclei were counterstained with 0.5 μg/mL Hoechst 33342, and cells were mounted in ProLong® Gold antifade reagent, dried overnight and sealed. The slides were then examined and imaged with a laser scanning confocal microscope (Zeiss, LSM710). A minimum of 100 acini were analyzed per group.