Immunocytochemistry Protocol for Cellular Protein Analysis

Immunocytochemistry analysis was performed according to a previous report^{21 (link)}. The cells were seeded onto slides. At 70% confluence, the cells were fixed in 4% paraformaldehyde in PBS buffer for 15 min. Following washes with PBS, the cells were permeabilized in freshly prepared 0.1% Triton X-100 for 10 min and blocked in 1% bovine serum albumin (BSA) in PBS buffer for 1 h at room temperature. The cells were incubated with primary antibodies directed against either runx2 (1:200, ab76956, Abcam), the α-subunit of BK (1:100, ab99046, Abcam, USA), integrin beta1 (1:100, 1798-1, Epitomics, USA), Flag (1:200, Rabbit Monoclonal Anti-Flag antibody, F2555, Sigma) or His (1:200, SAB1306084, Sigma) at 4 °C overnight. The secondary Alexa Fluor 647-conjugated anti-mouse antibody (Jackson Immuno Research, USA) was used at a 1:200 dilution for 2 h at room temperature protected from light. Following washes with PBS, the cells were stained with DAPI for 5 min. The slides were mounted with SlowFade™ Gold Antifade Mountant (S36936, Invitrogen), and images were captured using a Leica TCS SP5 confocal microscope (Leica, Germany) at room temperature with ×10 and ×40 objective lenses.