Target retrieval solution

Mice were perfused transcardially with 4% PFA/0.1 M PB. Brains were removed, immersed in the same fixative for 16–18 hours at 4°C, cut in the transverse plane (30 μm) and placed in cryoprotectant (30% ethylene glycol, 20% glycerol, 50 mM sodium phosphate buffer, pH 7.4) at −20°C until further processing. Sections were washed in sterile PBS, mounted onto charged slides, and dried overnight. Sections were incubated in hydrogen peroxide (10 min, 24°C), and then in target retrieval solution (5 min, 98–102°C; Advanced Cell Diagnostics, ACD, Hayward, CA), rinsed in sterile water (x 2), dehydrated in 100% EtOH (5 min), and dried before incubation in protease IV (30 min, 40°C). After rinsing in sterile water (x 2), sections were incubated with RNAscope® catalog oligonucleotide probes for Trpm4 and Nmb transcripts (2 h, 40°C), and processed according to manufacturer instructions provided in the Multiplex Fluorescent Reagent Kit v2 (ACD; RRID:SCR_012481). Slides were covered with Prolong Gold with DAPI anti-fade mounting medium (Molecular Probes, Eugene, OR).