Fv1100

For immunofluorescence staining, cells or tumor tissues from each treatment group were washed with ice-cold PBS and fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 20 min at room temperature, followed by permeabilization in 0.2% Triton X-100-PBS for 10 min. Samples were followed by blocking with PBS blocking buffer containing 2% normal goat serum, 2% BSA, and 0.2% gelatin for 1 h at room temperature. Then, the samples were incubated in primary antibodies at the appropriate concentration for 1 h at room temperature, washed with PBS and incubated in goat anti-rat-Alexa Fluor 647 (Molecular Probes) at 1:1000 dilution in blocking buffer for another 1 h at room temperature. Finally, stained cells were washed with PBS, counterstained with Hoechst 33342 (Molecular Probes-Invitrogen, H1399, 1:10000 dilution in PBS), and mounted on slides with Prolong Gold antifade mounting medium (Life Technologies). The slides were imaged under a confocal laser scanning microscope (Olympus, FV1100).

To assess the hG7-BM3 internalization in vitro, flow cytometry and confocal microscopy analyses were used in different cell lines. First, 5×10⁵ tumor cells (Huh-7/BEL-7402) were rinsed twice, split into eight experimental groups and incubated with 200 nM hG7-BM3 at 37°C. Timed groups (0, 2, 5, 15, 30, 40, 60, 90 min) were placed on ice and internalization was stopped. The flow cytometry assay followed the steps described above. The internalization percentage was calculated from the mean fluorescence intensities (MFI) as: % Internalization = [(MFI _TimeX-MFI_background) / (MFI_Time0 - MFI_background)] ×100.
We also used a fluorescence microscope to observe the internalization effect directly. hG7-BM3 was labeled with the visible fluorescent dye RhodamineB (Beyotime Institute of Biotechnology, Shanghai, China). The coupling method followed that described previously by Li Ding and the probes were named RhB-hG7-BM3. First, 5×10⁴ Huh-7/BEL-7402 cells were cultured overnight at 37°C and then incubated with 1 μM of RhB-hG7-BM3 for 2 h. After washing with PBS, the cells were imaged by a laser confocal microscope (Olympus FV1100). In addition, the free hG7-BM3 (50 μM) was mixed with RhB-hG7-BM3 (1 μM) to evaluate the competitive blocking [47 (link)].