FoxO1 and TH immunoflourescence was performed on free-floating brain or adrenal cryosections using FoxO1-specific and TH-specific monoclonal antibodies (Milipore Technology, FoxO1, Cat.No. 04-1005 and TH, Cat.No. AB152). Briefly, mice were perfused with 4% neutral buffered formalin and the brains/adrenals were dissected and post-fixed in 4% formalin overnight. After cryoprotecting in 20% sucrose overnight, the brains/adrenals were cryo-sectioned into 20-μm slides in cryotome. Sections were washed in PBS 1X (pH 7.4) 3 times for 10 min each and were permeated in 0.5% Triton X-100 in PBS 1X (v/v) for 30 min. After blocking step with 3% goat serum prepared in PBS 1X containing 0.25% Triton X-100 (PBT) for one hour at RT, the sections were then incubated at 4 °C for 48 h with anti-FoxO1 primary antibody (1:200 dilution) and for 24 h with anti-TH primary antibody (1:1,000 dilution) in PBT-azide containing 3% (v/v) goat serum. The sections were then washed with PBS 1X 4 times for 10 min each and incubated for 2 h in Alexa Fluor 488 goat anti-rabbit antibody (Invitrogen, CA, USA, Cat.No. A21206, 1:200 dilution) in PBT
