4.8. TUNEL Assay and Immunohistochemistry

Apoptotic cells in tumor tissue sections were detected using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay as previously described [44]. Irradiated tumor tissues were fixed with 10% neutral buffered formalin (NBF, Sigma-Aldrich, St. Louis, MO, USA) for 4 h and embedded in paraffin. After deparaffinization, TUNEL staining was performed using the In Situ Cell Death Detection Kit (Roche Diagnostics, Mannheim, Germany).

Immunohistochemistry (IHC) was performed as previously described [44]. Briefly, the tumor sections were sliced into 4 μm-thickness, deparaffinized in xylene, rehydrated in graded alcohol, and washed with 0.01 M PBS, pH 7.4. After heat-induced epitope retrieval with citrate buffer (pH 6.0; Dako, Carpinteria, CA, USA) and blocking with a blocking solution (Dako, Carpinteria, CA, USA), the tissue sections were incubated with anti-ATF4 rabbit polyclonal antibody (1:100; Abcam, Cambridge, UK) at 4 °C overnight. After washing with PBS, the samples were incubated for 30 min at room temperature with horseradish peroxidase-conjugated secondary antibodies (Dako, Carpinteria, CA, USA), and the slices were incubated with 3,3′-diaminobenzidine substrate chromogen solution (DAB, Dako, Carpinteria, CA, USA) for 5 min. Images for TUNEL and IHC were captured using an Aperio ScanScope AT slide scanner (Leica Biosystems Inc. Buffalo Grove, IL, USA) and analyzed using ImageScope software 12.4.3 (Leica Biosystems).