Anti cd34

Paraffin sections were deparaffinized and hydrated, and then rinsed three times with PBS, each time for 3 minutes. Next, the tissues were subjected to high-pressure antigen repair (Tris-EDTA PH9.0) for 2.5 min, treated with 3% endogenous catalase blocker (ZSBIO) for 15 min, and blocked using goat serum (ZSBIO). This was followed by incubation of the tissues with primary antibody at 37°C for 2-3 h and secondary antibody (ZSBIO) for one hour at room temperature. Tissues were washed with PBS and stained with DAB reagents (ZSBIO). Finally, the tissues were stained hematoxylin, differentiated with 1% hydrochloric acid alcohol, and sealed using neutral gum sealed. Results were evaluated by pathologists. The specific primary antibody used in IHC is anti-CD34 (1:1000, ZSBIO).

Clinical nasopharyngeal carcinoma samples and C57 BL/6 tumor xenografts were analyzed by the histochemical stain. Briefly, the paraffin-embedded sections of tumor tissues were deparaffinized and rehydrated. After antigen retrieval with 0.1M citrate buffer (pH 6.0), sections were blocked with 5% BSA for 30 minutes. The tumor samples were incubated with primary antibodies at 4°C overnight. Anti-EBV ZEBRA (BZLF1) (#sc-53904, Santa Cruz), anti-EBV EBNA1 (ab20870, Abcam), anti-CD34(#ZA-0550, ZSGB-bio), anti-CD163(#GB113152, Servicebio), anti-CD1a (#MA5-12526, Invitrogen) were used as the primary antibodies. PAS staining was performed using a PAS Staining Kit (#G1008, Servicebio). Images were acquired using Zeiss observer Z1, the numbers of classic angiogenesis were counted from three randomly chosen fields. Quantification of the positive area of CD1a versus CD163 was performed by the IHC Profiler function of IMAGE J.

Immunohistochemistry was performed according to the procedure described in our previous study (Zeng et al., 2012 (link)). Briefly, 4-μm-thick tissue sections were deparaffinized, rehydrated, and treated with an antigen retrieval solution (10 mmol/l sodium citrate buffer, pH 6.0). The sections were incubated with anti-TNC (1:250; Abcam) or anti-CD34 (1:100; ZSGB-BIO) overnight at 4°C and then incubated with biotinylated secondary antibody followed by addition of avidin-biotin peroxidase. Finally, tissue sections were incubated with 3′,3′-diaminobenzidine until a brown color developed, and they were counterstained with Harris' modified Hematoxylin. In negative controls primary antibodies were omitted. The evaluation of immunostaining was performed as previously described (Zeng et al., 2012 (link)). A score (ranging from 0–6) was obtained for each case. A combined staining score of≤2 was considered to be weak staining (no/low expression), a score between 3 and 4 was considered to be moderate staining (expression), and a score between 5 and 6 was considered to be strong staining (high expression).