Imaging Analysis of Mitochondrial Proteins and Amyloid in Prion-Treated Cells

N2a cells were cultured on 24-well coverslips and subsequently transfected with Mito-GFP, DsRed-Mito, Thioflavin T, or siRNA before treatment with PrP^106−126 or the control peptide. The specific processes were carried out in accordance with our previous studies (Song et al., 2022 (link)). Cells were washed twice with PBS before being fixed with 4% paraformaldehyde for 30 min. After washing twice with PBS, the cells were treated with immunostaining permeabilization buffer containing X-100 (Beyotime biotechnology, P0096) for 5–10 min at room temperature to permeabilize them. The cells were then blocked using an immunostaining blocking buffer (Beyotime Biotechnology, P0102) for 1 h at room temperature, followed by overnight incubation with specific primary antibodies (as described in the immunoblotting section) at 4°C (West et al., 2015 (link)). Following the PBS rinsing step, the cells were incubated with secondary antibodies for 1 h at 37°C and then washed with PBS five times for 5 min each. The coverslips were mounted on microscope glass slides using a fluorescent antifading buffer (Bioworld Technology, BD5014). Images were acquired using a Nikon A1HD25 confocal microscope at a magnification of 100 × (oil immersion lens). Approximately 10–15 unique images were captured at random for each sample. The acquired images were quantified using ImageJ software.