Mab318 af488

Sections were treated with 10 mM citrate (pH 6) of 65 °C at room temperature (RT) for 30 min. After three washes with PBS (5 min each), 10 mM glycine (in PBS) was applied for 20 min, followed by two washes with PBS (5 min). Sections were then permeabilized with 0.3% Triton X-100 for 30 min, washed twice with PBS (5 min) and pre-incubated in blocking buffer (SuperBlock®, ThermoFisher Scientific Prod# 37,515) for 30 min. Subsequently, they were incubated under gentle agitation at 4 °C overnight with each of the following primary antibodies: mouse monoclonal anti-KOR (1:25, 1:50) (ab201552, abcam), anti-Tyrosine hydroxylase Alexa Fluor 488 (1:100) (MAB318-af488, Millipore), rabbit anti-D4R (orb39453, Biorbyt), rabbit anti-D1R (orb107494, Biorbyt), mouse anti-D2R (1:600) (MABN53, Millipore) and rabbit monoclonal anti-SSTR2 (1:100) (ab134252, abcam). After two washes (5 min) with 1:2 dilution of blocking buffer in PBS, slices were incubated for 2 h at 37 °C with the secondary antibodies Alexa Fluor 488-conjugated goat anti-mouse (A11001) or anti-rabbit (A11034, A11008) (1:200, Invitrogen). Following two washes with the diluted blocking buffer in the dark, sections were mounted on SuperFrost glass slides with the addition of mounting medium (Duolink® In Situ Mounting Medium with DAPI, DUO82040, Sigma-Aldrich) and stored at − 20 °C.

Immunocytochemistry was performed as previously described [26 (link)]. Briefly, after fixing cells with 4% paraformaldehyde in phosphate buffered solution (PBS) (Cat. # AAJ19943K2, Thermo Fisher Scientific Inc.), they were permeabilized by adding cold methanol and then probed with the indicated primary antibodies. Antibodies used are as follows: anti-α-synuclein (#2642, 1:1000, Cell Signaling Technology), anti-phospho-α-synuclein (Ser129) (ab51253, 1:1000, Abcam plc.), anti-tyrosine hydroxylase (MAB318-AF488, 1:100, Millipore), anti-FOXA2 (ab108422, 1:300, Abcam plc), anti-phospho-IRS1 (Ser302) (Cat. # 2384, 1:1000, Cell Signaling Technology), and anti-IRS-1 (Cat. # 2382, 1:1000, Cell Signaling Technology). Fluorescence intensities from images of randomly selected microscopic fields of cells were semi-quantitatively analyzed based on staining positivity indices estimation using the NIH ImageJ software (https://imagej.nih.gov/ij/) as previously described by Jensen [27 (link)].