4.2. Immunocytochemistry (ICC)

In total, 1 × 10⁵ HER2-overexpressing breast cancer cells (SK-BR3, BT474, MDA-MB361) and HER2 low-expressing breast cancer cells (MDA-MB231, MDA-MB468) were seeded on coverslips at the bottoms of wells in a 24 well-plate. Cells were fixed in 4% paraformaldehyde for 15 min, washed with phosphate-buffered saline (PBS) twice, and non-specific sites were blocked with 3% bovine serum albumin (BSA) in PBS for 30 min at room temperature. Cells were incubated with anti-HER2 antibody (HER2/ErbB2 (D8F12) XP^TM Rabbit mAb, Cell Signaling technology, Danvers, MA, USA) overnight at 4 °C, followed by incubation with the appropriate Alexa Fluor 488 secondary antibody (1:1000, Anti-Rabbit IgG Fab2, Alexa Fluor (R) 488, Cell Signaling technology) for 1 h at room temperature. The cover slips were added the mounting medium with DAPI for 10 min prior to imaging by fluorescence microscope (LSM 700 confocal, ZEISS, Oberkochen, Germany).