We sought to verify previously labeled sections containing FISH probes for possible shell markers using adjacent tissue sections and immunohistochemistry to label for Calbindin. The sections were rinsed in Phosphate Tris (PT, pH 7.2-7.4) buffer and blocked in 10% normal donkey serum (Jackson, Cat# 017-000-121) solution for one hour. The sections were then incubated with primary antibody solution (Swant, Calbindin D-28K, 300, 1: 7,000) overnight at 4°C. The sections were then rinsed in PT buffer and incubated in secondary solution (Alexa Fluor 568, Donkey anti-mouse, 1:300) at room temperature for two hours. They were then rinsed in PT buffer and counterstained with Hoechst (Invitrogen, Cat# H21486, 1: 10,000) for 10 minutes. Lastly, the sections were rinsed in PT buffer and mounted with Prolong Gold Antifade (ThermoFisher, Cat# P36930). To verify μ-opioid receptor proteins were enriched in NUDAPs as well, we did immunostaining with MOR antibodies on an adjacent section performed with OPRM1 FISH labeling (Abcam, ab-10275, 1:500) using similar approach except that PBS buffer instead of PT buffer was used. To verify DRD2 and CPNE4 labels cholinergic neurons, after FISH
