Senescence associated (SA)‐β‐galactosidase staining

Fresh frozen OCT‐embedded tumor tissues were cut into 8 µm thick sections. The sections were fixed in 2% paraformaldehyde with 0.125% glutaraldehyde solution at room temperature for 5 min and rinsed with 1× PBS containing 2 mM MgCl₂. Thereafter, sections were incubated with X‐gal containing staining solution from senescence detection kit (Abcam ab65351) according to manufacturer’s instructions. The sections were counterstained with nuclear fast red (NFR) for 5 min. Slides were mounted in mounting media containing gelatin and glycerol. Brightfield images were acquired at 10× magnification using Nikon Ni‐E microscope with DS‐Ri2 camera. Multiple fields were imaged per tissue section from three tumor samples per treatment group. Quantifications were performed using Fiji ImageJ software. Briefly, color deconvolution was performed to segregate the SA‐β‐galactosidase (blue stain)‐positive tissue. Image threshold values were calculated and set to measure the percentage area covered by the entire tissue (NFR stained pink regions) and SA‐β‐galactosidase‐positive (blue stained) tissue. Percentage of SA‐β‐galactosidase‐positive tissue area was calculated, and individual values were plotted with GraphPad Prism. Statistical significance was calculated by Mann–Whitney unpaired 2‐tailed t‐test, and P‐value of < 0.05 was considered a significant difference.