Dtaf conjugated streptavidin

Locked-Nucleic-Acid (LNA) fluorescence in situ hybridization was performed as previously described (49 (link)) with modifications. Using RNase-free technique, mice were perfused with PBS, and freshly dissected lumbar cord tissue was embedded in OCT-Compond Tissu-Tek® (Sakura, Tokyo Japan). Lumbar cords were sectioned (10μm) on a cryostat (Thermo HM525) and then mounted on glass SuperFrost+ slides (Fisher Scientific). Slides were briefly fixed for 1 minute with freshly prepared 4% PFA. Slides were incubated in acetylation solution for 10 minutes, and then in hybridization solution at 60˚C for 4 hours. For hybridization, slides were hybridized with denatured DIG-labeled miR-124–3p or U6 RNA LNA probe (80 nM, Exiqon) overnight at 60˚C. Slides were then washed and incubated in blocking solution (1% BSA, 5% goat-serum, and 0.2% Triton-X 100 in PBS) for 1 hour. To amplify the LNA-probe signal, slides were incubated with HRP anti-DIG (1:1,000, 11207733910, Roche) for 1 hour, then incubated with biotin-tyramide (1:1,000, Sigma) for 1 hour, then incubated with DTAF-conjugated streptavidin (1:500, 016–010-084, Jackson ImmunoResearch) for 1 hour. The NeuN immunostaining (1:500, MAB377, Millipore) was performed following standard immunohistochemistry procedures. The slides were then mounted with ProLong Gold Anti-Fade mounting medium (Thermo Fisher Scientific).