Immunohistochemistry and Immunofluorescence Protocol

Immunohistochemistry and immunofluorescence were performed as reported (Sundaram et al., 2012 (link); Chen et al., 2017 (link)). In brief, mice were deeply anesthetized by 2% pentobarbital sodium and transcardially perfused with 4°C PBS and 4% paraformaldehyde. Brains were extracted and fixed in 4% paraformaldehyde for 24 h; 4 μm sections of the midbrain and the striatum were sliced by Leica RM vibratome (Leica Microsystems, Heidelberg, Germany). Slices for immunohistochemistry were incubated with anti-TH antibody (Millipore, Billerica, MA, United States). Goat against rabbit secondary antibody was from ZSGB-BIO (Beijing, China). Slices for immunofluorescence were incubated with anti-TH (Millipore, Billerica, MA, United States) or anti-GFP antibody (Aves Labs, Washington, DC, United States). Secondary antibodies were goat against rabbit antibody (Alexa Fluor^® 594) and goat against chicken antibody (Alexa Fluor^® 488). The nucleus was stained by DAPI (Boster, China). Images were obtained by research inverted system microscope (Olympus, Tokyo, Japan) or laser scanning confocal microscope (Olympus, Tokyo, Japan). The gray level was analyzed by ImageJ (National Institutes of Health, Bethesda, MD, United States) and the number of TH positive neurons was counted blindly by independent investigators.