Rnascope 2.5 universal pretreatment reagents

The frozen brain tissue was sliced into 10 μm coronal sections and mounted onto Colorfrost Plus slides (ThermoFisher, Waltham, MA, USA). Slices were incubated with hydrogen peroxide 10 min RT, target-retrieval solution and Protease III using RNAscope^® 2.5 Universal Pretreatment Reagents (Advanced Cell Diagnostics, #322380, Newark, CA, USA). smFISH for all genes examined, Rac1 (#517461), Arhgef6 (#574371), EGFP (#400281-C3), Sst (#404631-C2), Pvalb (#421931-C2), and Oprd1 (#427371-C3) were performed hybridization for 2 h. After hybridization, we used the RNAscope^® Multiplex Fluorescent Detection Kit v2 (#323110) to amplify signal and mounted. Images were acquired with a Nikon A1 microscope using 20× objective. IOD in SST⁺ or PV⁺ neurons was analyzed by Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD, US). The observer analyzing the expression of DOR, MOR,Rac1, and Arhgef6 in SST, or PV INs was blinded to the group allocation.

Mice brains were removed after perfusion, and 10-μm coronal sections were sliced from the frozen brain tissue and mounted on Colorfrost Plus microscope slides (Thermo Scientific, Waltham, USA). Slices were pretreated with hydrogen peroxide for 10 min at room temperature, and RNAscope 2.5 Universal Pretreatment Reagents (ACD: 322380, Advanced Cell Diagnostics, Newark, CA, USA) were used to perform target retrieval and proteolysis. In situ hybridization was then performed for 2 h at 40 °C with the probes of Egfp-C1 (ACD: 400281-C1) and Drd2-C2 (ACD: 406501-C2). After hybridization, RNAscope Multiplex Fluorescent Reagent Kit (ACD: 323110) was used to amplify the signal. After hybridization, the brain section was washed by 1 × wash buffer (PN 310091). Fluorescent images were captured by a Nikon-A1 confocal microscope with the 20 × air objective lens. The mRNA levels of Drd2 were analyzed by ImageJ (Fiji) (https://imagej.net/software/fiji/).