Immunocytochemistry of YAP

Cells were trypsinized and seeded at a concentration of 10 × 10⁴ onto frosted-coated glass slides and left to adhere overnight at 37°C in a 95% humidified 5% CO₂ incubator. Once adhered, slides were rinsed twice with sterile D-PBS and treated with 0.3% v/v Triton-X100 (T8787, Sigma) for five minutes to permeabilize the cells. Slides were washed three more times before blocking with 5% Goat serum (16210, Thermo Fisher) for 30 minutes before incubating with either primary YAP antibody (PA1-46189, Thermo Fisher) or 5% Goat serum (staining control) for 1 hour at room temperature. Slides were washed three times and treated with Goat Anti-Rabbit IgG Alexa Fluor^® 594 conjugated secondary antibody (A-11037, Thermo Fisher) for 60 minutes in the dark at room temperature. Slides were washed another three times and stained with ProlLong^® Gold Antifade Mountant with DAPI nucleus dye ({"type":"entrez-protein","attrs":{"text":"P36931","term_id":"2506707","term_text":"P36931"}}P36931, Thermo Fisher) for 10 minutes. Slides were mounted and used immediately for imaging and then stored at-20°C in the dark.