3.6. In situ hybridization

Brains from C57BL/6J mice were harvested and fixed as described for LacZ staining. Sections were cut at a thickness of 15 μm and mounted directly on slides then post‐fixed with 4% paraformaldehyde for 16 hr at 4°C.

Detection of transcripts in brain sections was performed using the RNAscope 2.5 HD Assay – BROWN kit (ACD). Tissue pretreatment was performed according to user manual no. 320534 and probe hybridization, counterstaining, and mounting of slides was performed according to user manual no. 322310‐USM. Slides were assayed using probes to either positive control (Ppib), negative control (dapb), or MCHR1 transcripts (ACD). Sections were counterstained with Hematoxylin, dehydrated, and mounted using Cytoseal (Thermo Scientific).