Cy3 conjugated goat anti rabbit igg

Vero E6 cells were seeded in 24-well plates and incubated at 37 °C overnight. HTNV, rVSV, or rVSV-HTNV-GP was used to infect cells for 2 h, followed by medium change to DMEM containing 2% FBS and cultured for an additional 48 h. Cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 15 min, permeabilized with 0.5% Triton X-100 for 10 min, and stained with 1A8 (HTNV NP) (1:1000), anti-Gn-1 (1:50), anti-Gc-10 (1:100), or anti-VSV-G (1:1000, abcam, 309106) antibodies at 4 °C overnight. After washing, Cy3-conjugated goat anti-mouse IgG (1:400, Sangon Biotech, D111024) or Cy3-conjugated goat anti-rabbit IgG (1:400, Sangon Biotech, D111018) were added to each well and incubated at room temperature for 1 h. The nuclei were visualized using Hoechst 33258 (1:1000, YEASEN, 40729ES10). The images were captured using a BX60 fluorescent cell imager (Olympus, Tokyo, Japan).

Female BALB/C nude mice bearing MDA-MB-231 tumors (approximately 100 mm³) were i.v. injected with PBS, Apo-Lf, or Holo-Lf (4 mg for each mice) for 24 h. The mice were i.v. injected with hypoxia probe pimonidazole hydrochloride (0.6 mg per 10 g body weight) 1.5 h before sacrifice. Subsequently, the tumors were cut into 10 µm sections and fixed with acetone. After washing with PBS twice, the slices were permeabilized in 0.5% Triton X-100 and blocked in 5% BSA and then incubated with anti-pimonidazole antibody (1:100, Hypoxyprobe) or anti-HIF-1α antibody (1:100, CST). After conjugation with corresponding fluorescence secondary antibodies, the slices were observed by CLSM. Also, MDA-MB-231 cells in different groups were incubated with the anti-γ-H2aX monoclonal antibody (1:400, CST) after 4 Gy exposure. The Cy3-conjugated goat anti-rabbit IgG was used as the secondary antibody (1:200, Sangon Biotech). After staining with DAPI, the cells were observed by CLSM. For γ-H2aX foci quantification, the total number of γ-H2aX foci in the nucleus of cells was counted. Analysis of 30 cells in 5 arbitrarily selected microscopic fields was conducted to determine the mean and standard error.