Cy3 goat anti rabbit igg h l

Immunofluorescence staining was performed on frozen sections. In brief, sections were rinsed in PBS and treated with 0.3 % Triton X-100 in PBS to disrupt the membranes. After blocking with 10 % normal goat serum for 1 h to reduce nonspecific staining, sections were incubated with the primary antibodies, mouse anti-beta III tubulin (Abcam, USA, 1:100) and rabbit anti-PGC-1α (Novus, USA, 1:150) overnight at 4 °C for 24 h. After rinsing in PBS, the secondary antibodies, Alexa Fluor 488 goat anti-mouse IgG (H + L) (Jackson, USA, 1:600) and Cy3 goat anti-rabbit IgG (H + L) (Jackson, USA, 1:800), were added onto sections in 5 % blocking solution, to incubate at room temperature for 30 min. Then, the sections were counterstained with DAPI (Sigma, USA), and imaged using a Leica DFC310 FX microscope (Leica, Japan).

After the indicated time post infection, the cells on glass slides were fixed with paraformaldehyde 4% for 10 min, washed with PBS, permeabilized with 0.5% Triton X-100 in PBS for 5 min (for H7 extracellular staining cells were not permeabilized) and incubated with antibodies against NP (catalogue number: PA5-32242; Thermo Fisher; 1:2000), H7 (ref. 44 (link)) (1:100), or M1 (provided by Dr. Jovan Pavlovic; 1:10) for 1 h at room temperature. After washing with PBS and incubation with corresponding secondary antibody Cy3 goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch; diluted 1:200), Alexa Fluor 488 donkey anti-chicken IgY (IgG) (H+L) (Jackson ImmunoResearch; diluted 1:500) or Cy3 goat anti-mouse IgG (H+L) (Jackson ImmunoResearch; diluted 1:200) for 30 min at room temperature, nuclei were stained for 5 min with DAPI (diluted 1:10,000 in PBS). The glass slides were examined on an Axioplan 2 Imaging System (Carl Zeiss, Oberkochen, Germany) equipped with an ApoTome.