Alexa fluor 488 conjugated goat anti guinea pig igg secondary antibody

The culture medium was removed, and then the cells were rinsed three times with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. After being washed with PBS three times and then blocked with 10% goat serum at 37°C for 1 h, the cells were incubated with a primary antibody against GLT-1 (1:500, #AB1783, Millipore, USA) overnight at 4°C. The cells were rinsed with PBS, exposed to 0.3% Triton X-100 at 37°C for 30 min and then incubated overnight at 4°C with a primary antibody against pAkt (1:1000, #ARG51558, Arigo). The coverslips were washed three times with PBS and then incubated with an Alexa Fluor 488-conjugated goat anti-guinea pig IgG secondary antibody (1:500, #A-11073, Invitrogen, USA) and a DyLight 594-conjugated goat anti-rabbit IgG secondary antibody (1:300, #072-09-15-06, KPL, USA) at 37°C for 60 min. Fluorescence images were captured by an investigator who was blinded to the experimental groups under a fluorescence microscope (Nikon Corporation, Tokyo, Japan).