Alexa fluor 488 conjugated goat anti mouse or anti rabbit igg

Herbal extract- or vehicle-treated, virus-infected cells on coverslips were fixed with cold acetone and methanol. The coverslips were blocked with normal horse serum (Invitrogen), and the cells' morphology was revealed by counterstaining with Evan's blue solution. The cells were then incubated with mouse monoclonal antibody against simian virus 40 (SV40) large T antigen (LT) (Calbiochem), which cross-reacts with BKPyV LT, or incubated with rabbit polyclonal antibody against JCPyV VP1 to detect the viral early or late protein, respectively. The primary antibody incubation was followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse or anti-rabbit IgG (Molecular Probes), respectively. Stained coverslips were mounted with antifade fluorescence mounting medium. Two thousand cells per coverslip were counted under a fluorescent microscope (Olympus) for determining the percentage of positive cells.

The following antibodies were used: mouse monoclonal anti-FLAG M2 (Sigma-Aldrich), mouse monoclonal anti-ubiquitinated protein (FK2; Nippon Bio-Test Laboratories), mouse monoclonal anti-Rab30 (Abcam, ab156774), rabbit monoclonal anti-Atg5 (Cell Signaling Technology, #4967), rabbit monoclonal anti-β-actin (Cell Signaling Technology, #4970), mouse monoclonal anti-LAMP1 (Santa Cruz, sc-20011), and rabbit monoclonal anti-GM130 (BD Transduction Laboratories, 610822). The secondary antibodies used for immunoblotting were horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (Jackson Immunoresearch Laboratories). The fluorescent secondary antibodies used for immunofluorescence were Alexa Fluor 488-conjugated goat anti-mouse or anti-rabbit IgG, Alexa Fluor 594-conjugated goat anti-mouse or anti-rabbit IgG, or Alexa Fluor 660-conjugated goat anti-mouse or anti-rabbit IgG (Molecular Probes/Invitrogen).