Dab substrate solution

The ability of recombinant baculoviruses to express antigenic HA protein of EIV H3N8 was further confirmed by immunoperoxidase staining of infected SF9 cells using the standard protocol of (Haegeman et al., 2020 (link)) with few modifications. In brief, Sf9 cells, cultured in six-well plates at 80% confluence, were infected with the recombinant baculoviruses at a multiplicity of infections (MOI) of 0.1. Following incubation at 27°C for 48 hours, the culture medium was discarded, and cells were washed twice with PBS, dried at 37°C for 1 hour, and frozen at −80°C for 30 minutes. Infected and control cells were fixed with 4% formaldehyde for 10 minutes and permeabilized with a mixture of methanol and hydrogen peroxide 30% (30:1) (Sigma-Aldrich). After removal of the fixatives and washing of cells twice with PBS, the equine anti-HA primary antibodies (diluted 1:256 in PBS) were incubated with cells at room temperature for 1 hour with gentle agitation. Cells were further washed three times with PBS-Tween, and HRP-conjugated anti-equine antibodies prepared in rabbit, diluted 1:1000 in PBS (Sigma-Aldrich) were added and incubated at room temperature for 1 hour. After another cycle of washing with PBS-Tween, cells were stained with DAB substrate solution (Sigma-Aldrich) and examined for immunostaining using the inverted microscope.