Alexa fluor 488 conjugated goat anti mouse igg

Cells were washed with PBS and fixed for 20 min with 4% paraformaldehyde at room temperature. After washing three times with PBS, the cells were permeabilized for 30 min with Immunostaining Permeabilization Solution with Saponin (Beyotime Biotechnology, Shanghai, China). The cells were then incubated with a monoclonal antibody against PRRSV-N protein (1:100) or dsRNA (1:200) overnight at 4°C. After being washed with PBS, the cells were incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (1:300) (Proteintech, Chicago, IL, USA) for 1 h at 37 °C. After an additional wash with PBS, fluorescence images were observed using fluorescence microscopy (Zeiss Vert. A1; Carl Zeiss Microscopy GmbH, Oberkohen, BW, Germany).

The following primary antibodies were used in our experiments: anti‐GAPDH antibody (Proteintech), anti‐Histone H3 antibody (Proteintech), anti‐phospho‐p70S6K antibody (T389) (Cell Signaling Technology), anti‐p70S6K antibody (Epitomics), anti‐LAMP1 antibody (Abcam), anti‐LAMP2 antibody (Santa Cruz), anti‐TFEB antibody (Cell Signaling Technology), anti‐p62 antibody (Enzo Life Sciences), anti‐mTOR antibody (Cell Signaling Technology), anti‐HA antibody (Santa Cruz), anti‐FLAG antibody (Sigma), anti‐GFP antibody (Santa Cruz), anti‐Rag B antibody (Cell Signaling Technology), anti‐raptor antibody (Cell Signaling Technology), anti‐α‐Tubulin (Proteintech), and anti‐C9orf72 (Santa Cruz). The following secondary antibodies were used: horseradish peroxidase‐conjugated sheep anti‐mouse and anti‐rabbit antibodies (Jackson ImmunoResearch Laboratories). The following fluorescent secondary antibodies were used: Alexa Fluor 594‐conjugated goat anti‐rabbit IgG (Proteintech), Alexa Fluor 594‐conjugated goat anti‐mouse IgG (Proteintech), Alexa Fluor 488‐conjugated goat anti‐rabbit IgG (Proteintech), Alexa Fluor 488‐conjugated Goat anti‐mouse IgG (Proteintech), Alexa Fluor 660‐conjugated goat anti‐mouse IgG (H + L) highly cross‐adsorbed antibody (Invitrogen), and Alexa Fluor 405‐conjugated goat anti‐rabbit IgG (H + L) secondary antibody (Invitrogen).

The slides of brain sections were fixed with 4% formaldehyde solution for 30 min at room temperature, incubated with 1% Triton X-100 for 30 min, and blocked with 5% goat or donkey serum for 1 h at 37 °C. Subsequently, sections were incubated overnight at 4 °C with the following primary antibodies: OTULIN (bs-14689R, Bioss Co., Beijing, China, 1:50), NeuN (MAB377, Millipore Co., Germany, 1:200), MAP2 (4542, Cell Signaling Technology, USA, 1:100) or Iba-1 (NB100-1028, Novus Co., USA, 1:200). After washing three times with PBS, sections were reacted with the following fluorescent secondary antibodies at 37 °C for 1 h: Alexa Fluor 594-conjugated goat anti-rabbit IgG (H+L; SA00006-4, Proteintech, 1:200), Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L; SA00006-1, Proteintech, 1:200), FITC-conjugated AffiniPure donkey anti-goat IgG (H+L; SA00003-3, Proteintech, 1:200), and 594-conjugated AffiniPure donkey anti-rabbit IgG (H+L; SA00006-8, Proteintech, 1:200). DAPI (Sigma, USA, 1:200) was used to stain cellular nuclei at 37 °C for 10 min. All images were observed and acquired using an A1+R laser confocal microscope (Nikon, Tokyo, Japan).