In Situ Hybridization Technique for Mouse Brain

In situ hybridisation (ISH) was carried out as previously described^{40 (link)}. Briefly, mice were perfused with 4% PFA in phosphate buffer using DEPC-treated water, and the brain tissue was dissected and post-fixed in the same buffer overnight. The tissue was frozen on the next day, and brain sections (20 μm) were prepared within a week. The sections were treated with 0.1 N HCl and Protease K, fixed, and acetylated. After pre-hybridisation in 50% formamide, 2x SSC, 1x Denhardt’s, 10 mM EDTA, 50 mg/ml tRNA, and 0.01% Tween20 at 55 °C for 1-2 h, the sections were hybridised with digoxigenin (DIG)-labelled RNA probes prepared using the DIG RNA labelling kit (Roche, #11175025910) in pre-hybridisation buffer supplemented with 5% dextran sulphate at 55 °C overnight. After RNaseH treatment to digest the unhybridised DIG-RNA, the brain sections were intensively washed with a low ionic buffer, and incubated with AP-conjugated anti-DIG antibody (Roche). The signal was visualised with NBT-BCIP. A fragment (1083–2036 nt) of the mouse Rfk gene (NM_019437) was cloned into pBluescript II and used as the template for probe synthesis.