Mounting medium with dapi h 1200

Immunocytochemistry was carried out as described previously. 30 In brief, bGCs were fixed with 4% formaldehyde diluted in warm phosphate-buffered saline (PBS) for 15 min. The primary Ab was YAP XP rabbit monoclonal Ab (mAb) (D8H1X; 14074, Cell Signaling Technology, Danvers, MA, USA) and the secondary Ab was anti-rabbit IgG (H+L), F(ab')2 fragment (Alexa Fluor 488 Conjugate; 4412, Cell Signaling Technology). The cells were incubated with YAP primary Ab (dilution 1:100) overnight at 4°C. After washing with PBS, the cells were incubated with the secondary Alexa Fluor 488conjugated Ab (dilution 1:1000) for 2 h at room temperature in the dark. Coverslipped slides were mounted with VECTASHIELD MOUNTING MEDIUM with DAPI (H-1200, Vector Laboratories, Burlingame, CA, USA), and images were taken with the Keyence BZ-X801 fluorescent microscope. I counted at least 50 YAP-stained cells from five randomly selected fields at 40× magnification, and calculated the proportion of nuclear YAP-positive cells to total counted cells.