Anti nap1l1

Immunohistochemical (IHC) staining was performed as described in previous study [16 (link)]. Briefly, Tissue sections prepared for antigen retrieval by microwave treatment in citrate buffer (pH 6.0) were incubated with anti-NAP1L1 (Sigma, America), anti-Ki67 (Zsbio, China), anti-cleaved-caspase 3 (Affinity, China) primary antibodies. Immunostaining was performed using the Envision System with diaminobenzidine (Dako Cytomation, Glostrup, Denmark). To assess the expression level of NAP1L1 in HCC tissue microarrays, a Vectra-Inform image analysis system (Perkin-Elmer Applied Biosystems) was used as described in previous studies [17 (link), 18 (link)].

Total protein were extracted using the Protein Extraction Kit (KeyGEN BioTECH, Nanjing, China) according to the manufacturer’s instructions. Protein lysates were separated by 10% SDS-PAGE and then transferred to a PVDF membrane. After the membranes were blocked, they were incubated with various antibodies at 4 °C overnight including, anti-GAPDH (CST, USA), anti-NAP1L1 (Sigma, Germany), anti-NOTCH1 (CST, USA), anti-OCT4 (Santa Cruz, CA, USA), anti-SOX2 (Santa Cruz, CA, USA), anti-c-MYC (CST, USA) and anti-ABCG2 (Abcam, UK). Then, the membranes were incubated with horseradish peroxidase-conjugated antibodies at room temperature for 45 min. Protein signals were detected using enhanced chemiluminescence (Pierce, Rockford, IL, USA).