Tyramide fitc kit

To synthesize digoxigenin-labeled (Roche, Penzberg, Germany) antisense RNA probes, we linearized pGEM-T easy-ccndx with PstI and transcribed it with T7 RNA polymerase. The probes for motor neuron progenitors and interneurons (isl1, olig2, nkx6.1, and vsx1) have been described previously27 (link)35 (link)36 (link)37 (link); these probes were linearized with NcoI and transcribed with SP6 RNA polymerase. To generate fluorescein-labeled (Roche, Penzberg, Germany) antisense probes, we linearized pcDNA3-CMV-GFP with HindIII and transcribed it with SP6 RNA polymerase. Whole-mount in situ hybridization was performed as previously described38 (link)39 (link). For double fluorescence in situ hybridization, the digoxigenin-labeled probes were detected using anti-digoxigenin POD (Roche, Penzberg, Germany) (1:500 dilution), and signals were amplified using a tyramide-FITC kit (Perkin Elmer, Boston, MA, USA). After the first staining step, the tyramide working solution was washed away and the embryos were incubated for 30 minutes with 1% H₂O₂, to inactivate the peroxidase activity of the first antibody. The embryos were then blocked for 1 hour, and the fluorescein-labeled probes were detected with anti-fluorescein POD (Roche, Penzberg, Germany) (1:500 dilution); the signals were subsequently amplified using a tyramide-Cy2 kit (Perkin Elmer, Boston, MA, USA).

After formaldehyde fixation (10% buffered formalin), brain tissues were paraffin embedded and sliced in 7 μm thick sections. DAB staining was performed as previously described [2 (link)]. Double immunofluorescence labelling was performed using Tyramide-FITC kit (NEL701A, Perkin Elmer), using a goat anti-rabbit antibody conjugated with biotin (Vector Laboratories, BA-1000) for SYNJ1 detection. Mouse monoclonal antibodies were detected using a goat anti-mouse antibody conjugated to Alexa 568 (A-11031, Invitrogen). Slides were mounted with Fluoromount-G (Southern Biotech) and immunofluorescence labelling was observed with an upright confocal microscope (Olympus Fluoview Fv1000) or with an Axiovert 200 M microscope (Zeiss) equipped with an ApoTome system (Zeiss). For quantitative analysis, SYNJ1 positive neurons and dystrophic neurites in hippocampal CA1–2 pyramidal layer were analysed at 40X images by thresholding analyses using NIH ImageJ as previously reported [58 (link)].