Free-floating sections were immunostained for TH or FOXP1 by overnight incubation in mouse anti- TH (Millipore, MAB-377, 1: 10,000 dilution) or rabbit anti- FOXP1 (Abcam, ab16645, 1: 50,000 dilution) primary in PBS with 0.25% Triton-X and 0.05% sodium azide. Afterwards, tissue was washed three times in PBS and incubated in biotinylated donkey-anti-mouse or anti-rabbit secondary (1:1000 dilution, Jackson Immunoresearch, West Grove, PA) for 30 min, followed by three 30 s rinses in PBS, followed by 1 hr in avidin-biotin complex (Vector). For TH-staining, tissue was then rinsed in sodium acetate buffer (0.1M, pH 7.4), followed by incubation for 5 min in 1% diaminobenzidine (DAB). For FOXP1 staining, nickel and hydrogen peroxide (Vector) were added to reveal a blue-black reaction product.
For florescent staining of FOXP1, free-floating sections were incubated in rabbit anti- FOXP1 (Abcam, ab16645, 1: 50,000 dilution) primary in PBS with 0.25% Triton-X and 0.05% sodium azide. Afterwards, tissue was washed three times in PBS and incubated in cy3-conjugated donkey-anti-rabbit secondary (1:1000 dilution, Jackson Immunoresearch, West Grove, PA).
For florescent staining of FOXP1, free-floating sections were incubated in rabbit anti- FOXP1 (Abcam, ab16645, 1: 50,000 dilution) primary in PBS with 0.25% Triton-X and 0.05% sodium azide. Afterwards, tissue was washed three times in PBS and incubated in cy3-conjugated donkey-anti-rabbit secondary (1:1000 dilution, Jackson Immunoresearch, West Grove, PA).