Bright otf5000 cryostat

Fresh frozen brains were collected from Nnat^+/+ and Nnat^+/−p male mice between 13 and 14 weeks of age. Coronal sections of 20-μm thickness were prepared on a cryostat (Bright OTF5000 cryostat) and mounted on RNase-free membrane-coated glass slides (Superfrost Plus, Thermo Fisher Scientific). Brain sections were fixed in 95% ethanol (30 s) and rehydrated in an ethanol series (75% ethanol, 50% ethanol) prior to staining with cresyl violet (Ambion). Sections were then dehydrated in another ethanol series of 50%, 75%, 95%, and 100% ethanol and left to air dry prior to laser capture. PVN and ARC, were then dissected using a PALM Microbeam Laser Capture Microdissection System (Zeiss) on a × 5 objective as previously described^{48 (link)}. Briefly, tissues were captured on AdhesiveCap Clear PCR tubes (Zeiss) and stored in 80 μl of QIAzol (QIAGEN) on dry ice prior to RNA extraction. Laser capture and subsequent cDNA library preparation were performed. RNA from laser-captured nuclei was extracted using the miRNeasy Micro RNA Extraction Kit (QIAGEN). Quality and quantity of the samples were then checked on an Agilent 2100 Bioanalyzer using Agilent RNA 600 pico and/or nano chips, with a typical sample RNA concentration in the range of 1–5 ng/μl.