Goat antimouse alexa fluor 488 labeled secondary antibody

To visualize the distribution of succinate receptor GPR91 on primary NSCs (that stain positive for nestin), we performed dual immunofluorescence staining. Briefly, primary NSCs were seeded on a coverslip and fixed with 4% paraformaldehyde. The coverslip was washed with phosphate-buffered saline, and the slides were treated with phosphate-buffered saline containing 0.1% Triton X-100 and 5% bovine serum albumin (Cat# VIC018, Vicmed, Xuzhou, China) for 1 hour. The slides were incubated with the following primary antibodies at 4°C overnight: rabbit anti-GPR91 polyclonal antibody (1:1000; Cat# 223051, United States Biological, Salem, MA, USA) and mouse anti-nestin monoclonal antibody (1:1000; Cat# NBP1-92717, RRID: AB_11020601, Novus Biologicals, Littleton, CO, USA). Then, the slides were incubated with goat anti-mouse Alexa Fluor 488-labeled secondary antibody (1:1000; Cat# A11001, RRID: AB_2534069, Thermo Fisher Scientific) or goat anti-rabbit Cy3-labelled secondary antibody (1:1000; Cat# AS007, RRID: AB_2769089, ABclonal, Woburn, MA, USA) at room temperature for 1 hour. Representative images were obtained using a laser-scanning confocal microscope (TCS SP5, Leica, Wetzlar, Germany). We used 4′,6-diamidino-2-phenylindole (10 mg/mL; Cat# KGA215-10, KeyGen BioTECH) to label cell nuclei.