cAMP measurements in primary striatal neurons
On day 8 of culture, where indicated cells were treated for 30 min with
SKF-38393 (100 µM), CCG-203920 (100 nM) and/or N/OFQ (0.01-1 nM).
Cultures were then fixed for 15 min in 4% paraformaldehyde. Following 3
× 5 min washes in 10 mM PBS-T (0.02% Triton X-100 in 10 mM PBS),
cultures were incubated in 5% bovine serum albumin (BSA) in 10 mM PBS-T
for 1 h at room temperature. Cultures were subsequently incubated in the
following primary antibodies: DARPP32 (R&D AF6259; 1:500), cAMP (R&D
MAB2146; 1:500), diluted in 1% BSA in 10 mM PBS at 4°C overnight.
Following 3 × 5 min washes in 10 mM PBS-T, cells were incubated in
594-conjugated secondary antibodies (Invitrogen; 1:500 A32814 or A11005)
in 1% BSA prior to 3 × 5 min washes. Cells were imaged using an Olympus
IX71 inverted microscope. The fluorescence intensity of individual cells
was measured by densitometry using Image J analysis software.