Gtx127357

To determine the expression of the HA, NA, and M1 proteins of the rBVs, Sf9 cells were infected with the indicated rBVs at a multiplicity of infection (MOI) of 1. Following 48 h of incubation at 27 °C, Sf9 cells were fixed with cold acetone for 10 min. The cells were then incubated with the primary antibodies, including rabbit polyclonal antibody against the H1N1 and H3N2 HA protein (1:500, GTX127357 and GTX127363, GeneTex, USA) and H3N2 NA protein (1:500, 40017-T60, Sino Biological, China), sheep polyclonal antibody against the H1N1 NA protein (1:500, AF4858, R&D, USA) or M1 proteins (1:500, ab22396, Abcam, USA) for 1 h, respectively. After washing with PBS, the secondary antibodies, Alexa Fluor 488-conjugated goat anti-mouse IgG, Alexa Fluor 594-conjugated donkey anti-sheep IgG or Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:500, ab150113, ab150180 and ab150080, Abcam, USA), were added to the cells and incubated at 37 °C for 1 h. The fluorescence signal was observed under a fluorescence microscope.

The HA protein expression in lung tissues was determined by multiplex immunofluorescent assay. Lung paraffin sections were deparaffinized in xylene and rehydrated in a series of graded alcohols. Antigen retrieval was performed in citrate buffer (pH = 6) by heating in a microwave for 20 min at 95 °C followed by a 20 min cool-down period at room temperature. Endogenous peroxidase was quenched with hydrogen peroxide for 20 min, followed by treatment with blocking reagent for 30 min at room temperature. The primary antibody (rabbit polyclonal antibody for H1N1-HA, GeneTex, GTX127357, 1:500) was incubated overnight in a humidified chamber at 4 °C, followed by detection using the Cy3-conjugated secondary antibody (Servicebio, GB21303, 1:300). The slices were imaged using confocal laser scanning microscopy.