Vibratome slices (thickness: 60 μm) were blocked for 2 hr with 5% Normal Donkey Serum in PBS, embedded in 4%
agarose (Sigma) and incubated overnight at 4°C with primary antibodies. Slices were then washed in PBS (3 3 20 min) and
incubated in secondary antibodies for 2 hr. Flat-mount preparations were frozen and thawed three times after cryoprotection (1
hr 10% sucrose in PBS, 1 hr 20% sucrose in PBS, and overnight 30% sucrose in PBS at 4°C), blocked with 5% Normal Donkey
Serum in PBS for 2 hr, and then incubated with primary antibodies for five days at 4○C and washed in PBS (3
× 1 hr). Subsequently, flat mounts were incubated with secondary antibodies for one day at 4○C and
washed in PBS (3 × 1 hr). The following primary antibodies were used in this study: goat anti-ChAT (1:1000, EMD
Millipore), mouse anti-PKAɑ (1:1000, Sigma),mouse anti-DDK (1:1000, Origene), rabbit anti-GFP (1:500, Invitrogen), and
mouse anti-GPR179 (1:1000, EDM Millipore). Secondary antibodies were Alexa 488- and Alexa 568 conjugates (1:1000,
Invitrogen).
agarose (Sigma) and incubated overnight at 4°C with primary antibodies. Slices were then washed in PBS (3 3 20 min) and
incubated in secondary antibodies for 2 hr. Flat-mount preparations were frozen and thawed three times after cryoprotection (1
hr 10% sucrose in PBS, 1 hr 20% sucrose in PBS, and overnight 30% sucrose in PBS at 4°C), blocked with 5% Normal Donkey
Serum in PBS for 2 hr, and then incubated with primary antibodies for five days at 4○C and washed in PBS (3
× 1 hr). Subsequently, flat mounts were incubated with secondary antibodies for one day at 4○C and
washed in PBS (3 × 1 hr). The following primary antibodies were used in this study: goat anti-ChAT (1:1000, EMD
Millipore), mouse anti-PKAɑ (1:1000, Sigma),mouse anti-DDK (1:1000, Origene), rabbit anti-GFP (1:500, Invitrogen), and
mouse anti-GPR179 (1:1000, EDM Millipore). Secondary antibodies were Alexa 488- and Alexa 568 conjugates (1:1000,
Invitrogen).