Immunofluorescence Staining of dsDNA and Hsp60

After treatment with IL6 or IL6+NAC, MFE-296 cells were fixed in 4% paraformaldehyde dissolved in PBS. Cells were permeabilized using 0.1% Triton X-100 on ice for 30 minutes; then, cells were blocked with 4% bovine serum for 1 hour at room temperature. Then, cells were incubated with primary anti-dsDNA (1 : 1000, Sigma) and anti-Hsp60 (1 : 1000, Proteintech), for 3 hours at room temperature, followed by incubation with FITC-labeled anti-IgG (1 : 500, Sigma) and Cy3-labeled secondary antibodies for an hour. Immunostaining images were collected using a confocal microscopy. Antibodies used to stain dsDNA have been validated in a previous study [20 (link)].