Anti eif4a

Immunohistochemistry was performed using anti-cleaved-caspase-3 (cat. no. 9664; Cell Signaling Technology, Inc.) and anti-eIF4A (cat. no. ab31217; Abcam) antibodies, as previously described (14 (link)). Two independent individuals examined the proportion of positive cells in ≥5 fields of view (magnification, ×400), and tissues were scored as follows: 0, negative; 1, <25% positive cells; 2, 25–50; 3, 50–75; and 4, >75%. Images were visualized and calculated using a Nikon microscope (Nikon Corporation). TUNEL assays was performed on paraffin-embedded tissue sections using a one-step TUNEL apoptosis assay kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Briefly, samples were incubated with TUNEL reaction mixture for 1 h at 37°C in the dark and then washed twice with PBS. Nuclei were counterstained with DAPI (300 nM; cat. no. D8417; Sigma-Aldrich) for 10 min at room temperature and mounting with antifade medium (cat. no. P0126; Beyotime Institute of Biotechnology) and then washed twice with PBS. The condensed or fragmented nuclei of apoptotic cells were observed using fluorescence microscopy (Olympus Corporation) (magnification, ×200) in 20 fields of vision.