Ab19853

IHC staining was performed with formalin‐fixed and paraffin‐embedded 196 HCC tissues sectioned to a thickness of 4 μm. The slides were placed in an ethylenediaminetetraacetic acid buffer for antigen repair. Forty microliters of diluted primary antibody (1:1000, ab19853; Abcam) was added to the slide and incubated for 30 min at room temperature. The slides were then treated with a secondary antibody for 60 min at room temperature. 3,3′‐Diaminobenzidine was used as the chromogenic substrate. Tumor tissues stained for the OS‐9 protein were scored using the histochemistry score, which was further used to transform the number of positive cells and their staining intensity in each section into corresponding values to achieve semi‐quantitative analysis. Finally, the median score was regarded as the cutoff and divided into high or low expression.

Equal amounts of protein samples were separated via sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore). After blocking with 5% blocking reagent for 1 hour, the membranes were incubated with primary antibodies against OS‐9 (1:1000, ab19853; Abcam), HIF‐1α (1:1000, ab179483; Abcam), and beta actin (1:5000, ab6276; Abcam) at 4°C overnight. After washing with Tris‐buffered saline containing 0.1% Tween 20, the membranes were incubated for 2 hours with horseradish peroxidase‐coupled anti‐rabbit immunoglobulin G (1:2500, ab6721; Abcam) at room temperature. Densitometry analysis of protein bands was performed using the Millipore ECL Western Blot Analysis Substrate (Merck Millipore). All bands were analyzed using the ImageJ software (Version 2.1.0; National Institutes of Health).