Anti neun antibody

For TUNEL staining of brain sections, the anti-NeuN antibody (Cell Signaling Technology #24307S, USA, 1:200) diluted in PBS was incubated with the sections overnight at 4°C. Goat anti-rabbit antibody 546 (red, Santa Cruz Biotechnology, USA, 1:200) were then biotinylated the tissue sections for 60 min. TUNEL staining was performed in accordance with the manufacture’s instructions of TUNEL system kit (Promega, USA). Image-pro Plus software (GE Healthcare, USA) was employed to analyze the number of neurons stained positively for TUNEL and NeuN in hippocampus and cortex by two persons blinded to the treatments.
For TUNEL staining of neuron, the cultured primary neurons were washed once with PBS, fixed with 4% paraformaldehyde at room temperature for 1 h and permeabilized using 0.1% Triton X-100. The anti-NeuN antibody (Cell Signaling Technology #24307S, USA, 1:200) was incubated with the fixed neuron overnight at 4°C. Then TUNEL reaction mixture were incubated with the fixed neurons at 37 °C for 1 h. DAPI was added to the wells for 5 min to stain nuclear after rinsing the cells with PBS. We counted the TUNEL-positive cells manually, and calculated the percentage of positive cells for each sample.

For motor neuron immunostaining and quantification, spinal cord sections from mice sacrificed at PD91 were used and prepared via slightly modified methods from our previous publications [24 (link),25 (link)]. In brief, 12–15 sections per mouse across 400 µm of the lumbar spinal cord were selected and incubated with primary rabbit monoclonal anti-NeuN antibody (1:500, Cat. #12943, Cell Signaling, Inc., Beverly, MA, USA) to label neurons. After incubation with secondary goat anti-rabbit Alexa Fluor 488 antibody (1:200, Cat. #111-545-045, Jackson Immunoresearch Laboratories, West Grove, PA, USA), neurons in the ventral horn were imaged using a Nikon Eclipse Ti epifluorescent microscope and Nikon NIS-Elements software (Nikon Instruments, Inc., Melville, NY, USA). Nuclei were labeled with Hoechst 33342 (bisbenzimide H 33342 trihydrochloride, Cat. #B2261, Sigma-Aldrich, St. Louis, MO, USA). For quantification of ventral horn MN, only large morphologically-intact neurons with a diameter ≥25 µm and distinct cell nucleus were counted per section. Small or injured neurons in the ventral horn with fragmented soma were not counted.

The level of autophagy was assessed according to a previous immunostaining protocol as follows: the slides of each coronal section were incubated in blocking buffer for 2 h, and then washed three times with PBS for 10 min. Afterward, incubated with anti-LC3 antibody (1:200; Novus Biological, Littleton, CO, USA; catalog number: NB600-1384), the slides kept overnight in a dark place at 4 °C. Subsequently, washed three times with PBS, the slides were incubated with anti-NeuN antibody (1:100; Cell Signaling Technology, Danvers, MA, USA; catalog number: 24307) under similar conditions. The following day, thoroughly washed three times with PBS, the slides were incubated with the corresponding secondary antibodies for 1 h at room temperature. After washing three times with PBS, the slides were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI; Beyotime Biotech Inc., Nantong, China; catalog number: C1002) for 2 min to show the locations of nuclei. Then, coverslips were used with the fluorescence quenching agent. We imaged the fluorescently stained cells via Olympus IX71 inverted microscope system and analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD).