H&E staining and immunohistochemistry were performed on both frozen and paraffin-embedded sections. Frozen tissues were first fixed in 4% paraformaldehyde for 24 hours, then transferred to a 30% sucrose solution for 24–48 hours, and finally snap-frozen in OCT compound (Sakura Finetek) using a dry ice and isopentane slurry. A cryostat was used to generate 10 μm tissue sections. Paraffin-embedded tissues were fixed in 10% neutralized formalin overnight and preserved in 70% ethanol until embedding in paraffin. Prior to staining, 5 μm tissue sections were deparaffinized in xylene and rehydrated in a graded series of ethanol and water washes.

For immunohistochemistry, endogenous peroxidase activity was blocked by treating sections with 3% H2O2, and antigens were retrieved with Antigen Unmasking Solution (Vector Laboratories). Tissues were blocked in 10% serum with 0.25% Tween 20 (MilliporeSigma) prior to incubation in primary antibody overnight. The following primary antibodies were used: rabbit polyclonal to HA-probe (1:250, Santa Cruz Biotechnology, sc-805), mouse monoclonal to Ser1981 phosphorylated ATM (1:200, MilliporeSigma, 05-740), rabbit anti–mouse Ser824 phosphorylated Kap1 (1:200, Bethyl Laboratories, A300-767A), and rabbit polyclonal to Ser15 phosphorylated p53 (1:200, Cell Signaling Technology, 9284). The tissues were incubated in biotinylated secondary antibodies for 30 minutes at room temperature and treated with VECTASTAIN Elite ABC Reagent (Vector Laboratories) per the manufacturer’s instructions. The DAB Peroxidase Substrate Kit (Vector Laboratories) was utilized to visualize positive staining prior to counterstaining with Mayer’s hematoxylin and dehydrating in a graded series of ethanol and water. Representative images were acquired with a Leica DFC450 bright-field microscope using Leica Suite software. ImageJ (NIH) was utilized to quantify positively stained cells or fractional areas by a single observer blinded to genotype and treatment.

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