Digital image analyzer

In brief, the tissue sections were deparaffinized in xylenes and rehydrated through graded (100%–95%–70%) ethanols to distilled water. Then, sodium citrate buffer (0.01 M) was used for antigen retrieved with high pressure conditions for 15 min. After that, samples were washed with phosphate-buffered saline (PBS; 0.02 M) for 3 minutes three times at room temperature. Subsequently, samples were incubated with the rabbit anti-TRIM11 (10851-1-AP, Proteintech, USA), anti-DUSP6 (ab220811, Abcam, UK), and anti-ERK1/2 (ab184699, Abcam, UK) antibodies in PBS overnight at 4 °C followed by Alexa Fluor 488 goat anti-rabbit IgG (H + L) (A0423, Beyotime, China) for 1 hour at room temperature. Images were acquired by an ECLIPSE Ni microscope and a digital image analyzer (NIKON, Japan). Three replicates were needed for each analysis.

Cells were fixed with 4% paraformaldehyde for 30 min at room temperature. After that, samples were washed with PBS three times, for 3 min each time, at 25°C and blocked with 1% BSA (Solarbio, Beijing, China) for 1 h at room temperature. Subsequently, cells were incubated with rabbit anti-γ-H2AX antibody (ab2893, Abcam, UK) in PBS overnight at 4°C followed by goat anti-rabbit IgG (H+L) (A0423, Beyotime, Haimen, China) for 1 h at room temperature. Images were obtained by an ECLIPSE Ni microscope and a digital image analyzer (Nikon, Tokyo, Japan).