Ki67 d3b5

Tissues were fixed overnight in Mildform 20N (Wako, Osaka, Japan), embedded in paraffin, and sectioned at 4 μm thickness. Sections were immersed in sodium citrate (pH 6.0) buffer for antigen retrieval, and subsequently incubated at 4 °C overnight with primary antibodies as follows; SFXN1 (12296-1-AP, 1:400; ProteinTech, Rosemont, IL), Ki-67 (D3B5, 1:400; Cell Signaling Technology, Danvers, MA) and cleaved caspase-3 (5A1E, 1:2000; Cell Signaling Technology). They were probed with anti-rabbit IgG antibody labelled with Histofine Simple Stain MAX-PO (Nichirei Bioscience, Tokyo, Japan) and visualized with diaminobenzidine (Wako). The TUNEL assay was conducted using DeadEnd Colorimetric TUNEL System (Promega, Madison, WI) according to the manufacture’s protocol. Nuclei were stained with hematoxylin. The intensity score of SFXN1 staining was determined in HCC tissues and adjacent liver tissues of each sample, ranging from 0 to 4. Tumor samples with a score of 0 or 1 and with a score of 2, 3 or 4 were categorized into the low and high expression groups, respectively.

Tissues were fixed overnight in 4% paraformaldehyde, embedded in paraffin, and sectioned (4 μm thick). Sections were immersed in sodium citrate (pH 6.0) buffer for antigen retrieval, and subsequently incubated with primary antibodies against CD8 (D4W2Z, 1:500; Cell Signaling Technology), glutamine synthetase (ab64613, 1:100; Abcam) and Ki-67 (D3B5, 1:200; Cell Signaling Technology) at 4 °C overnight. They were probed with anti-mouse or anti-rabbit IgG antibody labelled with peroxidase Histofine Simple Stain MAX-PO (Nichirei Bioscience, Tokyo, Japan), and visualized with diaminobenzidine. Nuclei were stained with hematoxylin.