Vectashield hardset antifade reagent with dapi

Two to 6 weeks after viral injections of Cre-dependent PPO into the VPM, mice were deeply anesthetized with ketamine/xylazine and transcardially perfused with PBS followed by 4% PFA. Brains were removed and cut at a 45° angle to the right of the midline to preserve thalamocortical axon projections in the plane of slicing. They were then post-fixed in PFA overnight, cryoprotected in 30% sucrose and frozen in OCT media. 50 μm thick thalamocortical slices were then cut parallel to the 45° plane and stored in PBS with 0.2% NaN₃. Sections were rinsed thoroughly in PBS before being blocked and permeabilized in PBS with 2% donkey serum, 2% goat serum and 0.3% Triton X-100 for 1 hour at room temperature. Primary antibodies (mouse anti-PSD-95 (1:500) and guinea pig anti-vGluT2 (1:500)) were incubated with the sections overnight at 4°C in PBS with 2% each donkey and goat serum. Sections were washed 5 times and incubated with secondary antibodies (donkey anti-mouse AF555 (1:500) and goat anti-guinea pig AF647 (1:500)) for 1 hour at room temperature in PBS with 2% donkey and goat serum. Sections were washed 5 times again and mounted onto slides using Vectashield Hardset Antifade reagent with DAPI (Vector Labs, cat. # H-1400). Thalamocortical slice imaging was performed on an Andor Dragonfly microscope described below.

Following behavioral experiments, mice were deeply anesthetized with pentobarbital and transcardially perfused with PBS followed by 10% formalin. Heads were placed with optic fibers intact overnight in 10% formalin. Brains were extracted and post-fixed in formalin overnight, followed by cryoprotection in 30% sucrose. 30 μm thick slices containing either the VTA, BLA or NAc core, shell were collected and mounted onto slides using Vectashield Hardset Antifade reagent with DAPI (Vector Labs, cat #H-1400) and imaged using an Olympus FV1000 confocal microscope (Olympus Scientific Solutions).