Anti rat igg hrp

For western blot analysis, an equal volume of the EV fractions were mixed with Laemmli buffer (Bio-Rad Laboratories) and boiled at 95°C for 5 minutes. Samples were then loaded on a 10% SDS-PAGE gel and transferred onto PVDF membrane (Bio-Rad Laboratories). The membranes were blocked with 5% non-fat milk dissolved in PBS for 1 h and then probed with primary antibodies overnight at 4°C (primary antibodies: anti-CD9 (1:100, Abcam), anti-Flotillin (1:500, Cell Signaling), anti-Alix (1:100, Santa Cruz Biotechnology), anti-Calnexin (1:500, Abcam), anti-GM130 (1:250, BD Biosciences), anti-CD81 (1:100, Santa Cruz Biotechnology), anti-CD63 (1:100, BD Pharmingen), anti-APOA1 (1:500, Santa Cruz Biotechnology). After 4 washes in PBS containing 0.1% Tween (PBST), membranes were incubated with corresponding HRP-conjugated secondary antibodies: anti-mouse IgG-HRP (1:1000, Santa Cruz Biotechnology), anti-rabbit IgG-HRP (1:1000, Thermo Fisher Scientific) or anti-rat IgG-HRP (1:1000, Thermo Fisher Scientific) for 1h and washed in PBST. Signals were visualized after incubation with Amersham ECL Prime substrate and imaged using an ImageQuant LAS 4000.