Nbp2 30221

Immunohistochemistry was performed as previously described (32 (link)). Briefly, paraffin embedded tissue slides with human HCC tissue microarray (TMA) (NBP2-30221, Novus Biologicals) were deparaffinized and rehydrated, endogenous peroxidise activity was blocked with 3% hydrogen peroxide, antigen retrieval was performed in 10 mmol/L citrate buffer, and nonspecific binding was blocked with blocking reagent. HAVCR2 (ab185703, Abcam) and C10ORF54 (CL3975, Invitrogen) antibodies were applied at 1:300 and 1:20 concentrations, respectively. Slides were incubated overnight at 4°C, followed by 30 min incubation with secondary anti-mouse or rabbit antibody HRP (Dako). The chromogen used was 3-amino-9-ethylcarbazole. Human normal and cancerous lung tissue was used as the positive control for both the antibodies and a negative control, for which the primary antibodies were substituted with the same concentration of mouse or rabbit IgG. Images were captured using a Olympus CX41 microscope and QCapture software. Immunohistochemical reactivity was evaluated by two independent investigators. The expression of HAVCR2 and C10ORF54 were categorized into positive staining or no staining.

Immunohistochemistry was performed with the standard protocol [46 (link)]. Briefly, paraffin-embedded tissue slides of human HCC tissue microarray (TMA) (NBP2-30221, Novus Biologicals) were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide, and antigen retrieval was performed in 10 mM citrate buffer. The primary antibodies for NFE2L1 (#8052, Cell Signaling Technology) and STX12 (PA5-59407, Thermo Fisher Scientific) were used. The signals were developed with a diaminobenzidine (DAB) substrate solution (Thermo Fisher Scientific), and nuclei were counter-stained with hematoxylin. Images were captured using the slide scanner AxioScan. ZI (Zeiss, Oberkochen, Germany). DAB-stained intensities were graded 0 (no staining), 1 (weak), 2 (moderate), or 3 (strong) for NFE2L1 and 0 (no staining), 1 (weak), or 2 (moderate) for STX12.