Fitc labeled goat anti mouse igg

The Sf9 cells cultured in 12-well plates were infected with the four recombinant baculoviruses (Ac-Cap, Ac-Cap-T, Ac-Cap-B and Ac-Cap-TB) at an MOI of 1. After 48 h post-infection, the cells were fixed using cold methanol/acetone (1:1) for 30 min at −20 °C and then blocked with 2% bovine serum albumin for 1 h at room temperature. The cells were incubated with anti-Cap MAb (1:100 dilution) for 1 h at 37 °C and then washed with PBS for three times. Subsequently, the cells were incubated with fluorescein isothiocyanate (FITC)-labeled-goat anti-mouse IgG (1:100 dilution, ABclonal) for 1 h at 37 °C and then washed with PBS for three times. The cells were analyzed using a confocal microscope (LSM 510, Carl Zeiss, Heidenheim, Germany).

Ferrous iron (Fe²⁺) was measured using an iron assay kit (Sigma) and a FerroOrange probe (Dojindo Laboratory). For the iron assay kit, iron is released by the addition of an acidic buffer and then reacted with a chromogen, resulting in a colorimetric product (593 nm), which is directly proportional to the Fe²⁺ concentration. The absorbance at 593 nm was measured using a plate reader (Bio-Rad). The FerroOrange probe was used for fluorescence imaging of intracellular Fe²⁺ (54 (link)). Briefly, FerroOrange (1 mM) dispersed in serum-free medium was added to the cells, followed by incubation for 30 min at 37°C. The cells were then fixed with 4% paraformaldehyde for 45 min and permeabilized with 0.2% Triton X-100 for 20 min. After that, the cells were blocked with phosphate-buffered saline (PBS) containing 5% bovine serum albumin (BSA) for 1 h and then incubated with anti-HSV-1 gD antibody (1:1,000 in 5% BSA) overnight, followed by staining with FITC-labeled goat anti-mouse IgG (ABclonal) (1:1,000 in 5% BSA). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime) for 5 min at 37°C in the dark. Cells were photographed under a confocal microscope (A1R; Nikon, Japan). The fluorescence intensity was analyzed using ImageJ software.