Alexa fluor 488 conjugated polyclonal goat anti mouse secondary antibody

A total of 2 × 10⁶ cells were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 15 min at room temperature in the dark. The cells were washed and incubated with 1% BSA for 1 h at room temperature. After incubation, the cells were washed with PBS and incubated with 0.5 nM anti-EGFR (199.125; Thermo Scientific, Rockford, IL, USA) for 3 h at room temperature. The cells were then incubated with Alexa fluor 488-conjugated polyclonal goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, Inc., USA) for 1 h at room temperature. After washing with PBS, fluorescence was detected with BD FACS LSRII SORP system (Becton Dickinson Company, New Jersey, USA), and confocal microscopy was performed using an LSM 700 instrument (Carl Zeiss, Oberkochen, Germany).

Formalin-fixed, paraffin-embedded tissue samples were sectioned into serial 4-mm slices and placed on microscope slides. After deparaffinization in xylene and rehydration in alcohol, tissue sections underwent antigen retrieval for 30 min in Tris-EDTA buffer titrated to pH 9.0 at 97°C and cooled with tap water. Non-specific antigen reactions were blocked by a 1-h incubation with M.O.M. mouse IgG blocking buffer. The slides were then incubated with the anti-EGFR (MA5-13319; Thermo Scientific, Rockford, IL, USA) at a dilution of 1:500 for 12–16 h at 4°C. The slides were then incubated with Alexa fluor 488-conjugated polyclonal goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, Inc., USA) at a dilution of 1:100 for 1 h at room temperature. After washing with PBS, the slides were examined under confocal microscopy using LSM 700 instrument (Carl Zeiss, Oberkochen, Germany).