Goat anti mouse igg h l horseradish peroxidase conjugate

SK-L cells were inoculated with the serially 10-fold diluted CSFV in 96-well plates and incubated at 37 °C for 72 to 96 h. Then, the plates were immunostained as described previously [27 (link)]. Briefly, the plate was air-dried and heat-fixed at 80 °C for 1 h. Cells were then incubated at room temperature for 1 h in the presence of the primary monoclonal antibody for NS3, 46/1 [28 (link)]. After washing with PBS, cells were incubated at 37 °C for 1 h in the presence of goat anti-mouse IgG (H + L) horseradish peroxidase conjugate (Bio-Rad, Hercules, CA, USA). The primary and secondary antibodies were diluted 2000-fold with PBS containing 1% Bovine Serum Albumin Fraction V (Roche, Basel, Switzerland) and 0.05% Tween 20 (FUJIFILM Wako Pure Chemical Corp.). Finally, cells were washed again with PBS and then stained with 3-amino-9-ethyl carbazole (Sigma-Aldrich). Virus titers were calculated and expressed as TCID₅₀ per milliliter [29 (link)].

The immunoperoxidase assay was performed as previously described [40 (link)]. Briefly, cells inoculated with viruses were washed with PBS and heat-fixed at 80 °C for 1 h. The cells were then incubated at room temperature for 1 h in the presence of the primary monoclonal antibody for NS3, 46/1. The cells were washed with PBS and then incubated at 37 °C for 1 h in the presence of goat anti-mouse IgG (H+L) horseradish peroxidase conjugate (Bio-Rad). The cells were washed again and then stained with 3-amino-9-ethyl carbazole (MilliporeSigma).