Primary rabbit anti his tag mab

SW480 cell lines were seeded on coverslips and cultured in dishes at 37 °C with 5% CO 2 , when 80% confluent cells were formed. 20 μM recombinant antibody RGD4C-scFv was added, and incubated for 5 h at 37 °C. And then fixed with 4% paraformaldehyde for 30 min. After permeabilized with PBS containing 0.2% Triton X-100 (Sigma-Aldrich, Darmstadt, Germany) and washed with PBS containing 0.02% Tween-20 (PBST)threetimes, the slides were incubated overnight at 4 °C with primary rabbit anti-His Tag mAb (clone number: D3I1O, Cell Signaling TECHNOLOGY, USA) and mouse pan-Ras mAb (clone number: C4, SANTA CRUZ, USA), washed for 5 min with PBS then incubated for 1 h at 37 °C in the dark with FITC-conjugated goat antirabbit antibody (ZSGB-BIO) and TRITC-conjugated goat anti-mouse antibody (ZSGB-BIO). Nuclei were stained with 4′,6-diamidino-2-phenylindole (Sigma, Da, Germany) at 25 °C for approximately 15-20 min. The fluorescence signals were analyzed with a fluorescence microscope (OlympusBX51, Tokyo, Japan).