Immunofluorescence Staining of Caco-2 Cells

Caco-2 cells were fixed using 4% Paraformaldehyde (PFA) in cytoskeleton stabilisation buffer (10mM PIPES at pH 6.8, 100mM KCl, 300mM sucrose, 2mM EGTA and 2mM MgCl₂) for 20 min at room temperature (RT) or were fixed using 100% methanol for 10 min at −20°C. Staining for Myosin VI, DP, keratin-8, keratin-18, Dsg2, tagRFPT and GFP required pre-permeabilisation of cells (0.1% Triton in PBS) for 3 min on ice before fixation with PFA. Subsequently, the fixed cells were blocked in 3% Bovine Serum Albumin (BSA) in PBS at RT for 1 hour. Following this, cells were incubated with primary antibody for 1 hour at RT. For Myosin VI and Dsg2 primary antibodies, the cells were incubated overnight at 4°C. Then, the cells were washed with 0.05% Tween in PBS before incubating with the corresponding secondary antibodies at RT for 1 hour. Following this, the coverslips were mounted using Prolong gold (Genesearch #8916BC) with or without DAPI.