Double immunofluorescence staining

Double immunofluorescence staining for Beclin-1 or LC-3 (autophagy related proteins) with NeuN (neuronal staining specific marker) was performed to count cells and neurons undergoing autophagy (Toepfer et al., 2011; Han et al., 2018). The specific method has been described in previous studies (Liu et al., 2014; Cao et al., 2017). Briefly, the rat brain samples were fixed in 4% paraformaldehyde for 24 hours at 4°C, then dehydrated in a 30% sucrose solution. Brain samples were sectioned at a thickness of 10 µm. Double immunofluorescence staining for Beclin-1 with NeuN was similar among the groups. The primary antibodies were rabbit anti NeuN polyclonal antibody (1:200, rabbit polyclonal, ab128886; Abcam, Cambridge, UK), rabbit anti LC-3 polyclonal antibody (1:300, ab48394; Abcam) and rabbit anti Beclin-1 polyclonal antibody (1:500, ab62557; Abcam), and were diluted in PBS overnight at 4°C. The sections were washed with PBS and incubated with goat anti-rabbit IgG secondary monoclonal antibody (1:500; Beyotime, Shanghai, China) at room temperature for 1 hour. The sections were incubated and covered with DAPI, rinsed again and covered with glycerol. The results were observed and analyzed by fluorescence microscopy (Leica Microsystems, Wetzlar, Germany).