A primer used for sequencing was 5′-CCC TCA TAG TTA GCG TAA CG-3′ (-96 gIII sequencing primer, provided in the see more Ph.D.-12 Phage display peptide library kit). Homologous analysis and multiple sequence alignment
were done using the BLAST and Entospletinib price Clustal W programs to determine the groups of related peptides. Cell-Based ELISA with Phage A498 and HK-2 were cultured in DMEM with 10% FCS at 37°C in a humidified atmosphere containing 5% CO2, and the cells were seeded into 96-well plates (1 × 105 cells/well) overnight. Cells were then fixed on 96-well plates by 4% paraformaldehyde for 15 min at room temperature until cells were attached to the plates. Triplicate determinations were done at each data point. Selectivity was determined using a formula as follows : Selectivity = ODM13 – ODC1/ODS2 – ODC2. Here, ODM13 and OD C1 represent the OD values from the selected phages and control phages binding to A498 cells, respectively. OD S2 and ODC2 represent the OD values from the selected phage and control phage binding to the control (HK-2 cell line), respectively. Immunocytochemical Staining and Immunohistochemical Staining of Phage M13 Before staining with phage M13 , the cells in the different groups (A498 and HK-2) were cultured on coverslips and fixed with
acetone at 4°C for 20 min. Then, about 1 × 1011 selleck kinase inhibitor pfu of phage M13 diluted in PBS were added onto the coverslips and incubated at 4°C overnight. Coverslips
were then washed for five times with TBST. The coverslips were blocked by H2O2 (3% in PBS) at room temperature for 510 min. After being washed by PBS for 5 min at 37°C, the coverslips were incubated with normal sheep serum for 20 min at 37°C. Subsequently, the coverslips were incubated overnight at 4°C with a mouse anti-M13 phage antibody at a dilution of 1:5000. The next day, the coverslips were rinsed selleck compound for three times (10 min for each rinse) in PBS and incubated with a secondary antibody for 1 h at room temperature. Afterward, the coverslips were rinsed three times (5 min for each rinse) in PBS. The bound antibody was visualized using DAB. The coverslips were rinsed for three times (5 min for each rinse) using running tap water before staining by hematoxylin and eosin. Finally, the coverslips were rinsed for 10 min with running tap water before dehydration and mounting. Frozen sections of human renal tissues with and without tumors were also prepared. The steps of immunohistochemical staining were similar to those for immunocytochemical staining described above. Instead of the selected phage clone M13, PBS and a nonspecific control phage with same titers were used for negative controls. The study protocol was reviewed and approved by the Institutional Review Board and Ethic Committee of the First Affiliated Hospital of Sun Yat-Sen University (NO.