2.6. miRNA in situ hybridization

In situ hybridization of miRNA was performed using a modified in situ protocol (²³) with DIG‐labeled miRCURY LNA™ detection probes (QIAGEN) for miR‐219 (miRCURY LNA Detection probe #619109‐360) and Scramble‐miR negative control (miRCURY LNA Detection Control probe #699004‐360). Paraffin‐embedded mouse brain sections (10 μm) were deparaffinized and rehydrate, and then incubated with 10 μg/ml of proteinase K (P4850, Sigma‐Aldrich) at 37°C for 10 min. Washed sections with PBS were incubated in imidazole buffer [0.13 M 1‐methylimidazole (Wako Chemicals), 300 mM NaCl, pH 8.0], followed by an incubation in EDC‐Imidazole solution [0.16 M 1‐ethyl‐3‐(3–dimethylaminopropyl) carbodiimide (Tokyo Chemicals, Tokyo, Japan) in imidazole buffer, pH 8.0] for 2 h at room temperature. After washing the sections, 25 nM DIG‐labeled probe in G‐Hybo‐L hybridization buffer (GenoStuff, Tokyo, Japan) was hybridized to each section overnight. The hybridization temperature = DNA Tm probe − 20–21°C. For probe detection, washed sections were incubated with alkaline phosphatase‐conjugated anti‐DIG antibody (1:1,000, Roche, Basel, Switzerland) in Blocking buffer [1% Blocking reagent (Roche), 150 mM NaCl, 100 mM Tris‐Cl, pH7.5] overnight at 4°C. Washed sections with TBST (50 mM Tris‐HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.5) were soaked in Staining Buffer (100 mM NaCl, 100 mM Tris‐HCl, 50 mM MgCl₂, pH9.5), followed by an incubation with NBT/BCIP solution (1:50, Roche) in Staining Buffer at room temperature. Slides were then washed in PBS and water, and mounted for color bright‐field microscopic observations using a Keyence BZ‐9000 microscope equipped with a 20 × objective. miR‐219‐positive (miR‐219⁺) cells were counted in the region between 3 and 4 mm anterior to lambda as follows: for the corpus callosum, the counted cells were within the coronal section 1 mm from the midline, whereas for the cortical gray matter, the cells were counted within the parasagittal section 1 to 2 mm from the midline. The cells were assessed in at least four microscopic visual fields in one tissue section using five brain sections per animal. Four animals were analyzed for each genotype.