Cd31 antibody

Slides containing tissue sections were deparaffinized with xylene and rehydrated with ethanol. Then, antigen retrieval was performed by placing the slides in Tris/EDTA buffer (Solarbio Life Science, Beijing, China) before heating for 10 minutes. The slides were then treated with endogenous peroxidase blocker for 10 minutes and normal 10% goat serum (Solarbio Life Science, Beijing, China) was utilized for 30 minutes to block nonspecific binding sites. Different antibodies were applied to the slides and incubated overnight at 4 °C. After the hybridization of secondary antibodies, DAPI was used to stain the cell nucleus. The slides were observed using a fluorescence microscope (OLYMPUS, Tokyo, Japan). Antibodies against γ-H2AX, p16, p21 and p53 were obtained from Abclonal Biotechnology (Wuhan, China); CD31 antibody was purchased from Bioworld Technology (Nanjing, China); eNOS antibody was obtained from Abcam Biotechnology (Cambridge, UK). α-SMA antibody was purchased from Affinity (OH, USA).

Tumor tissues were fixed with 10% neutral formaldehyde solution, embedded in paraffin and cut into 3–4 μm thick sections for immunohistochemical analysis. Ki-67 antibody (Bioworld technology Co. Ltd. Nanjing, China), CD-31 antibody (Bioworld technology Co. Ltd.) and in situ cell death detection kit (Roche, Mannheim, Germany) were used to detect Ki-67 expression, MVD and apoptosis, respectively. All staining steps were carried out according to the manufacturer's instructions. Images were taken by a microscope.
The proliferation index was quantified as the percentage of Ki-67-positively stained cells (brown cell nucleus), while apoptotic number was quantified as the number of TUNEL-positive cells (brown cell nucleus). MVD was evaluated according to the method described by Weidner et al [30 (link)]. MVD value was obtained as the mean number of CD-31-positive tubular structures in the five most vascular fields under high-power field (magnification ×400) per section.

EJ cells, HEK 293 cells, pcDNA3.1(−)-myc-RI, pGEX-4 T-RI and pEGFP-C1-RI plasmids were conserved and prepared by our laboratory. pGEX-4 T-1 was bought from GE Healthcare China. pEYFP-N1vector was from Clontech. pCMV-3xflag-CMVTM-10 was purchased from Sigma. BALB/C nude (nu/nu) mice were obtained from Beijing HFK Bioscience Company (Beijing, PR China). FBS was from TBD Science (Tianjin, PR, China). DMEM/High glucose medium, RPMI 1640 medium and G418 were purchased from Gibco-BRL (Carlsbad, CA, USA). Lipofectamine 2000 was bought from Invitrogen, Inc., (Carlsbad, California). Monoclonal mouse antibody of anti-human ILK was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-human β-actin, CD31 antibody, Monoclonal primary rabbit antibody of anti-human PI3K, p-PI3K, PTEN, p- PTEN, Akt, p-Akt, GSK3β, p-GSK3β, mTOR, p-mTOR and β-catenin were obtained from Bioworld Technology, Inc. (St. Louis, USA). The rest of the primary antibodies are from Proteintech Group, Inc (Chicago, IL, USA). Cell Counting Kit-8 was bought from Genview Scientific, Inc (Craigieburn, VIC, AUS).