Alexa fluor 555 conjugated donkey anti rabbit

Marc-145 cells plated onto a cover glass in a 24-well plate were transfected with pVAX-mVIP or pVAX-1. Twenty hours later, the cells were infected with PRRSV BB0907 (MOI = 1) and incubated for 36 h. After being fixed with 1:1 methanol/acetone, the cells were incubated with mouse anti-PRRSV N, GP5 (made in our laboratory) and rabbit anti-FLAG-mViperin antibodies (Cell Signaling), mouse anti-calnexin (Cell Signaling) for 1 h at 37°C. After being washed three times, the cells were incubated with a mixture of Alexa Fluor 555-conjugated donkey anti-rabbit (Beyotime) and FITC-conjugated goat anti-mouse secondary antibodies (Boster) for 1 h at 37°C. The nuclei were stained using DAPI, and cover slips were mounted onto a slide glass using 10% glycerol. After three washes, confocal images were obtained using a Zeiss LSM 710 scanning confocal microscope.

Anti-tyrosine hydroxylase (TH) (ab112, Abcam, Cambridge, UK); Anti-TH (sc-25,269, Santa Cruz, Santa Cruz, USA); anti-cleaved Caspase 3 (9664; CST, Danvers, USA); Anti-Fyn (ab184276, Abcam, Cambridge, UK); anti-p-Y416 (6943; CST, Danvers, USA); anti-mTOR (2972; CST, Danvers, USA), anti-p-mTOR (Ser2448) (5536; CST, Danvers, USA); anti-p70 S6 Kinase (9202; CST, Danvers, USA); anti-p-S6K(Thr389) (97,596; CST, Danvers, USA); anti-β-actin (20536–1-AP, Proteintech, Wuhan, China), FITC-conjugated goat anti-rabbit IgG (Proteintech, Wuhan, China); FITC-conjugated donkey anti-mouse (Proteintech, Wuhan, China); Alexa Fluor 555-conjugated donkey anti-rabbit (Beyotime, Haimen, China); PP1 (HY-13084,MCE,New Jersey, USA); rapamycin (HY-10219, MCE, New Jersey, USA); all-trans retinoic acid (RA) (R2625, Sigma–Aldrich, Missouri, USA); glucose (15,023,021,Thermofisher, Waltham, USA).