Alexa 633 anti rabbit

All animals were deeply anesthetized with ketamine-xylazine and transcardially perfused first with cold PBS (20mL) followed by 30–40 mL of cold 4% PFA (paraformaldehyde, diluted in PBS), and brains were post-fixed in 4% PFA overnight at 4°C. For cryoprotection they were next put in 30% sucrose (diluted in PBS) overnight and frozen at - 45°C in isopentane. The brains were cut with a freezing microtome (Thermo Fisher), with nominal section thickness set to 20 μm. After rinsing with PBS, slices where incubated 2h at room temperature in 0.3% PBT (0.3% Triton X- in PBS) and 10% BSA blocking solution. Primary antibodies diluted in 0.3% PBT and 0.1% NGS (normal goat serum) were incubated overnight at 4°C. The following antibodies were used: anti-CB1 (Frontiers institute, goat 1:400), anti-GFP (Millipore, MAB 3580 mouse 1:500), anti-DsRed (Clontech, rabbit 1:500), anti-PV (Sigma PARV-19, mouse 1:1000) and anti-SST (Santa Cruz G10 sc-55565, mouse 1:250). Slices were then rinsed with PBS and incubated for 2h at room temperature with the secondary antibodies Alexa 488 anti-goat, Alexa 488 anti-mouse and Alexa 633 anti-rabbit, all obtained from Life Technologies and diluted 1:500 in PBT 0.3%. Slices were mounted with DAPI Fluoromount (Sigma) and stocked at 4°C. Whole brain slices were imaged using an epifluorescence slice scanner (Axio scan Z1 Zeiss, magnification 20×).