Cy3 conjugated goat anti rabbit

The cells were fixed with 4% PFA for 30 minutes, then the cells/tissue sections were washed with PBS for 5 minutes three times at room temperature. The frozen tissue sections were treated with formaldehyde at 0, 3, 6, 12 hours, 1, 3 and 7 days after modeling, then blocked with blocking buffer (Beyotime, Shanghai, China) for 1 hour. Next, the tissue sections were treated with primary antibodies [Ezh2, 1:200, rabbit monoclonal antibody (CST, USA); NF160/200, 1:500, mouse Monoclonal antibody (Sigma-Aldrich); IB4, 1:200, FITC conjugate (Sigma-Aldrich); TuJ1, 1:1 000, mouse Monoclonal antibody (Sigma-Aldrich); NeuN, 1: 300, rabbit monoclonal antibody] at 4°C overnight. Eighteen hours later, tissue sections were incubated with secondary antibodies [Cy3-conjugated goat anti-rabbit, 1:500 (Sigma); 488-conjugated goat anti-mouse, 1:500 (Sigma-Aldrich)] for 1.5 hours at room temperature, and then mounted on microscope slides. Finally, an antifade mounting medium with 4,6-diamino-2-phenyl indole (DAPI) solution (Beyotime) was used to seal the slice. Samples were visualized and images captured using an optical and epi-fluorescence microscope (Axio Imager M2, Carl Zeiss Microscopy GmbH, Jena, Germany). All assays were performed in triplicate.