NPC biopsies, validated by pathologist Dr. Desheng Xiao (Xiangya Hospital), were obtained from Pathology Department of Xiangya Hospital. The NPC tissue array was purchased from Pantomics (Richmond, CA, USA). Paraffin sections from NPC patient samples were firstly dewaxed and antigen retrieved in citrate buffer using a microwave for 15min. After cooling of the citrate buffer to room temperature, the sections were incubated with PBS (containing 5% bovine serum albumin (BSA), and 3% FBS) for 30 min, and subsequently incubated with HoxB3 (ab83404, Abcam), HoxB13 (ab53931, Abcam), HoxC8 (HPA028911, Sigma) or LMP1 (M0897, DAKO) primary antibody for 1 h. The slides were thoroughly washed three times with PBS (5%BSA, 3%FBS) solution for 10 min each and then incubated for 30 min with HRP- conjugated secondary antibody for 30 min at room temperature. The slides were thoroughly washed three times with PBS before using DAB. The images were surveyed and captured using a CX41 microscope (OLYMPUS, Tokyo, Japan) with the Microscope Digital Camera System DP-72 (OLYMPUS, Tokyo, Japan) and differentially quantified by two pathologists, Dr. Bo Li and Dr. Songqing Fan (The Second Xiangya Hospital, Changsha, China).
In situ hydridization (ISH) was performed using the EBERs HRP conjugated probe and DAB as substrate from ISH kit (Life technologies), according to the instructions of the manufacturers.
In situ hydridization (ISH) was performed using the EBERs HRP conjugated probe and DAB as substrate from ISH kit (Life technologies), according to the instructions of the manufacturers.