Immunocytochemistry

hNCC line #1 cells were plated on coverslips pretreated with ECL Cell Attachment Matrix; the next day, media was changed for serum-free with GH for 24 h then cells were fixed with 4% PFA/PBS for 20 min. Cells were permeabilized with 0.25% Triton X-100 for 30 min and blocked in 10% donkey serum in 1% BSA/PBS. Anti-WIP1 mouse primary antibodies and anti-HIPK2 rabbit antibodies diluted in 10% donkey serum were added overnight, followed by secondary donkey anti-mouse Alexa Fluor 488, anti-rabbit Alexa Fluor 568 antibodies, and Alexa Fluor 647 phalloidin in 1% BSA/PBS for 1 h. Samples were imaged with a Stellaris confocal microscope (Leica Microsystems).