P0102

Spread the cells in a 6-well plate with glass slides until the cell confluence reaches 70%, fix them with 4% paraformaldehyde for 30 min, seal them with immunofluorescence sealing solution (Beyotime, P0102) at room temperature for 2 h, then incubate them with primary antibodies (Supplemental Table 2) overnight at 4 °C. On the second day, incubate with second antibody (1 : 1000) for 1 h, then stain with DAPI for 5 min, wash off with PBS, and photograph under a microscope.

After being treated as described in method 2.6, cells were washed using 0.5% PBST and fixed with paraformaldehyde of 4% for 10 min, followed by being washed using 0.5% PBST three times and blocked using immunol staining blocking buffer (P0102, Beyotime Institute of Biotechnology, Shanghai, China) for 1.2 h. Then the cells were incubated with Nrf2 rabbit anti-human primary antibody (1:100 dilution) at 4°C overnight, followed by being kept at room temperature for 1 h and washed using 0.5% PBST three times. The cells were subsequently incubated with FITC labeled goat anti-rabbit secondary antibody (1:200 dilution) at 37°C in a humidified incubator for 1.5 h, followed by being washed using 0.5% PBST three times. Finally, cells were stained with Hoechst 33258 of 20 μM for 5 min, followed by being washed using 0.5% PBST three times and adding antifade polyvinylpyrrolidone mounting medium (P0123, Beyotime Institute of Biotechnology, Shanghai, China). Then the slices were observed via a fluorescence microscope (Leica DMI400B).

Frozen tumor sections were fixed and immersed in sodium citrate buffer to retrieve the antigen. The sections were then treated with anhydrous methanol containing 0.3% H₂O₂, and blocked with an immunostaining blocking solution (Beyotime; P0102) for 1 hour at room temperature. After sequential incubation with the primary and secondary antibodies (Gene Tech; GK500705), the sections were incubated in DAB chromogenic solution for 2 min and counterstained with hematoxylin for 5 min.

The brains tissues of mice were fixed in 4% paraformaldehyde buffered with PBS at 4^°C for 24 h, followed by changing into 30% sucrose solution for another 24 h. After embedding by optimum cutting temperature (O.C.T.) compound (Solarbio, Shanghai, China), the midbrain was cut into 16-μm-thick serial brain sections containing the SNpc for the subsequent immunofluorescence staining. The slices were incubated with blocking solution (P0102, Beyotime, Beijing, China) including BSA and Triton X-100 for 1 h followed by the incubation overnight at 4^°C with anti-tyrosine hydroxylase (TH, ab112, Abcam, Cambridge, MA, USA, 1:500) and anti-ionized calcium-binding adapter molecule 1 (Iba-1, ab178847, Abcam, 1:200) antibodies, respectively. The primary antibodies were diluted by QuickBlock™ Primary Antibody Dilution Buffer (P0262, Beyotime). Then the slices were incubated with secondary fluorescent antibodies fluorescein isothiocyanate (FITC)-labeled Goat Anti-Rabbit IgG (green, A0562, Beyotime, 1:1,000) for 2 h. The secondary antibodies were diluted by QuickBlock™ Secondary Antibody Dilution Buffer (P0265, Beyotime). After that, fluorescence microscope (Carl Zeiss, Oberkochen, Germany) were applied to photograph.

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.

Cells were seeded on sterilized coverslips in a 12-well plate and allowed to adhere and categorized into different experimental groups according to specific treatment conditions. Subsequently, cells were gently washed with PBS buffer and fixed with 4% paraformaldehyde for 15 min at room temperature. Permeabilization was achieved with 0.1% Triton X-100 for 20 min and non-specific binding was minimized by incubating the cells in a blocking buffer (P0096 and P0102, Beyotime, China) for 30 min. Cells were incubated with a primary antibody specific to the target protein in a humidified chamber at 37 °C for 60 min. Afterward, cells were washed with PBS buffer to remove unbound primary antibodies and subsequently incubated with anti-Rabbit conjugated to Alexa Fluor 555 or anti-Mouse conjugated to Alexa Fluor 488 (M213411M and M21011M, Abmart, China) at a 1:200 dilution in a humidified chamber at room temperature for 60 min. The coverslip was inverted onto a slide with an Antifade Mounting Medium with DAPI (P0131, Beyotime, China). Using a confocal fluorescence microscope (Olympus, FV3000, Japan) to observe and record.

Briefly, the procedure for IF staining was as follows: tissue sections were dewaxed with xylene, ethanol, and distilled water, and the sections were further subjected to antigen collection and permeation with 0.3% Triton-X 100, followed by blocking with immunol staining blocking buffer (P0102, Beyotime, Shanghai, China). Then, the sections were incubated with the primary antibodies overnight at 4 °C; after incubation, sections were again incubated with the corresponding secondary antibody. The antibodies used in this manuscript were glial fibrillary acidic protein (GFAP, 1:50, ab7260, Abcam, Cambridge, UK), ionized calcium-binding adaptor molecule 1 (Iba-1, 1:50, ab178847, Abcam), Neuron (NeuN, 1:100, 66836-1-Ig, Proteintech, Wuhan, China), and NLRP3 (1:100, 27458-1-AP, Proteintech). After 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) staining, the result was observed by fluorescence microscope (BX 53 Olympus, Tokyo, Japan).