Ab36367

Immunofluorescence and immunochemistry were performed as previously described (Cosset et al., 2015 (link), 2016 (link)). The following primary antibodies were used: mouse anti-neuronal nuclei-specific protein (NeuN) (Chemicon;MAB377), rabbit anti-βIII-tubulin (Covance;PRB435P), goat anti-ChaT (Chemicon; AB144P), rabbit anti-HB-9 (Abcam; ab922606), rabbit anti-ISLET1 (Abcam; ab22450), goat anti-GALR3 (Abcam), mouse anti-EV-71 (Abcam; ab36367), and mouse anti-PV-3 (Abcam; ab22450). Alexa Fluor (555 and 488)-labeled antibodies from goat or donkey against mouse, goat, or rabbit (Molecular Probes) were used as secondary antibodies. Cell nuclei were stained with DAPI (4, 6-diamidino-2-phenylindole). For IHC, biotin-conjugated anti-rabbit IgG or anti-goat IgG were used and developed using avidin-biotin peroxidase detection system (Vector Labs) with 3,3′-diaminobenzidine substrate (DAB, Sigma-Aldrich) after 2 min of incubation.

HeLa cells were grown in F10mm coverslips in 24-well plates overnight. Plasmids expressing pEGFP-PMLIII, pEGFP-PMLIV, and the empty plasmid pEGFP-C1 were transfected into the cells with Lipofectamine 3000. 24 h post transfection, the cells were infected with EV71 (MOI = 5) for 24 h. The cells were then washed three times with PBS before being fixed with 4% paraformaldehyde for 15 min at room temperature and then permeabilized with 0.1% Triton X-100 for 10 min. The slides were washed three times with 0.1% PBS-Tween, blocked with blocking buffer (4% dry milk without fat), stained with a mouse anti-VP1 antibody (ab36367, Abcam), and followed with a goat-anti-mouse-Alexa 594 (1:1,000 dilution). Nuclei were stained with DAPI at 1 μg/ml (Sigma-Aldrich). Fluorescent images were acquired using an Olympus FluoView FV10i (Tokyo, Japan) confocal microscope.

Immunofluorescence was performed as previously described (Fu et al., 2017 (link)). Antibodies used for staining are as followed: anti-VP1 antibody (ab36367, Abcam), Flag (F1804, Sigma), DDX6 (A300-460A, Belthy-Lab). The secondary antibodies are: Alexa Fluor goat anti-mouse-594 IgG (h+L) (1:1000 dilution), Alexa Fluor goat anti-rabbit-594 IgG (h+L) (1:1000 dilution) and Alexa Fluor goat anti-rabbit-488 IgG (h+L) (1:1000 dilution, Life Technologies). All images were taken using the Olympus FV3000 confocal microscope (Tokyo, Japan).