Sc 271625

Antigen retrieval was performed using a high-pressure method in 0.01 M sodium citrate-hydrochloric acid buffer (pH = 6.0) on tissue sections for 2 min, followed by immersion in antigen retrieval solution (C1035, Solarbio Science & Technology, China) for 5 min and blocking with 5% goat serum containing 0.3% Triton X-100 for 1 hour at room temperature. Then, the sections were incubated with the following antibodies at 4 °C overnight: rabbit anti-OPTN (1:200, 10837-1-AP, Proteintech Group), mouse anti-TDP-43 (1:200, sc-376311, Santa Cruz), mouse anti-LC3β (1:200, sc-271625, Santa Cruz) and rabbit anti-NEUN (1:50, ab177487, Abcam). After the primary antibodies, the sections were incubated with fluorescein isothiocyanate-AffiniPure goat anti-rabbit IgG (1:250, 111-095-003, Jackson) and Red-X-AffiniPure goat anti-mouse IgG (1:250, 115-295-003, Jackson) antibodies, and the cell nuclei were counterstained with DAPI (C1005; Beyotime Co., Shanghai, China) at room temperature for 3 min and placed in anti-fade fluorescence mounting medium. All images were acquired by fluorescence microscopy (Olympus Corporation, Tokyo, Japan), and Pearson’s correlation coefficient (Rr) and Manders’ overlap coefficient (R) were calculated by Image-Pro Plus software to analyse the colocalization. NeuN-positive cells were counted by ImageJ from 24 images. Four mice from each group were collected.