Immunofluorescence Imaging of Cellular Stress Response

Approximately 1 × 10⁴ cells were seeded in 8-chamber Slides (Corning) 24 h after siRNA or plasmid transfection. Cells were treated with ABS (pH 4, 200 μM) for 20 min and recovered for designated time points. After the treatment, cells were fixed with fresh-made 4% formaldehyde for 45 min. Then immunofluorescence staining was performed as previously described [35 (link)]. Antibodies for IGFBP2 (Cell signaling), EGFR (Invitrogen) and Phospho-DNA-PKcs (Thr2609) (Invitrogen) were incubated at 4 °C for overnight. Alexa fluor-568 conjugated anti-rabbit and Alexa fluor-488 conjugated anti-mouse secondary antibody (Invitrogen) were incubated for 1 h at room temperature. Cells were mounted with DAPI solution. Images were captured using the All-in-one Fluorescence Microscope (BZ-X700) (Keyence, Itasca, IL, USA).