Dab chromogen

A 100 μL cell suspension was aliquoted to prepare cell smears, and the cells were fixed. Subsequently, the cell smears were flushed thrice with PBS and blocked with 1% BSA. Then cell smears were incubated with the mouse anti-TNFAIP2 antibody (Santa Cruz, USA, 1:200) at 4° C overnight. Then the cell smears were sequentially incubated with the secondary antibody and the DAB chromogen (Gene Tech, USA). Finally, the cell smears were stained with haematoxylin (Baso, Zhuhai, China), sealed with neutral resin.

IHC was performed using standard techniques. Briefly, 5-µm paraffin-embedded TMA sections were dewaxed using xylene (Sinopharm, China)and rehydrated in graded alcohols (Sinopharm, China). Next, blocked endogenous peroxidase with 3% hydrogen peroxide (sinopharm, China). Antigen retrieval was accomplished via adding 10 mM citrate buffer (pH 6.0) (Sinopharm, China) to the TMA sections and putting them into a high-pressure cooker. Then, the TMA sections were incubated with 1% bovine serum albumin (BSA; Sigma, German) for 20 min to reduce nonspecific protein binding. The recombinant rabbit monoclonal CDC45 antibody (1:50; HuaBio, Hangzhou, China) were next used to treat the TMA sections at room temperature for 1 h. They were then rinsed with phosphate-buffered saline (PBS; HuaBio, Hangzhou, China) and incubated with biotinylated secondary antibody (MXB, Fuzhou, China) for 30 min at room temperature. Subsequently, the TMA sections were stained with DAB chromogen (Gene Tech, Shanghai, China), counterstained with Mayer’s haematoxylin (HuaBio, Hangzhou, China), dehydrated with gradient alcohol and xylene, and mounted on slides. Finally, the TMA sections were viewed under a Nikon light microscope.

A gradient ethanol series was used to rehydrate paraffin-embedded tissue sections from NPC patients and mouse xenografts for IHC staining. Endogenous peroxidases were blocked by hydrogen peroxide (0.3%). The sections were blocked with goat serum for 30 min, incubated with primary antibodies overnight at 4 °C, and finally incubated with secondary antibodies for 1 h at room temperature. DAB chromogen (Gene Tech, Shanghai, China) was used to stain the sections, which were subsequently counterstained with hematoxylin. According to the manufacturer’s instructions, dewaxed sections were stained with hematoxylin and eosin for HE staining.