In situ hybridisation

Sections were incubated in Xylol for 2 hours and then 5 min in 99.9%, 96% and 70% ethanol respectively. Sections were rinsed with TBST (TBS + 0.1% Tween 20) and then incubated with Proteinase K (10 μg/mL) for 7 min at 37 °C. After washing the sections with TBS they were treated with 4% PFA for 10 min. Sections were washed and treated with 0.2% Glycin in TBS, rinsed again with TBS and acetylated for 30 min with triethanolamin/acetic anhydrid. Slides were then rinsed in hybridization buffer (5× SSC, 50% formamide and 5× Denhardt’s, 250 μg/ml yeast t-RNA (Sigma) 2% blocking reagent (Roche), 0.1% Tween 20) for 2 h at RT. Probes (4 pmol) were incubated in hybridization buffer overnight at Tm −20 °C (Tm provided by Exiqon) in a humidified chamber. miR-124 served as positive control for hybridization and miR-scr served as negative control. All probes were 5′-3′-digoxigenin-labeled. The following day sections were washed in 5× SSC to remove cover slides and then washed with washing buffer 1 (50% formamide, 1xSSC, 0.1% Tween 20), for 30 min at Tm −20 °C. Then sections were rinsed in washing buffer 2 (0.2 × SSC) for 15 min at room temperature and TBST for 5 min. Sections were incubated with blocking solution (for 1 h at RT and then incubated with Anti-DIG-Fab POD antibody (1:200, Roche) for 1 h at RT. After rinsing sections with TBST they were treated with TSAPlus Cy3 working solution (PerkinElmer) for 10 min. Afterwards sections were washed again with TBS. As control sections were co-stained with anti-GFAP antibody (Millipore) as follows: after blocking (2% BSA, 0.1% Tween 20) sections were incubated with anti-GFAP (Millipore) overnight. The following day sections were washed in PBS and incubated in blocking solution for 20 min at RT. Sections were incubated with Alexa Fluor 488 labelled antibody (Life technologies) for 2 h and washed with PBS.