Histological analysis, IHC, and immunofluorescence

For IHC or immunofluorescence staining, the brain sections were dehydrated with a gradient post fixation. As described in our previous study [³²], the sections encompassing the entire SNpc were cut into 30-μm-thick slices with a cryostat (Leica, Model M1950–1–0-0-1-1). Then, the sections were incubated with 0.3% Triton X-100 (Sigma, T9284) in phosphate-buffered saline (135 mM NaCl, 4.7 mM KCl, 10 mM Na₂HPO₄, 2 mM NaH₂PO₄, pH 7.4) for 15 min (and then treated with 3% hydrogen peroxide, if needed) and blocked with 5% goat serum (Life Technology, 16,210–064) for 1 h at room temperature. Next, the sections were incubated with the following primary antibodies at 4°C overnight: anti-TH (Sigma, T1299; 1:800), anti-GFAP (Millipore, MAB360; 1:800), anti-AIF1/IBA1 (Wako, 019–1974; 1:1000), anti-ITGAM/CD11b (AbD Serotec, MCA275R; 1:250), anti-NLRP3 (AdipoGen, AG-20B-0014-C100; 1:500), anti-ubiquitin (Abcam, ab7780; 1:250), anti-MAP1LC3B (Cell Signaling Technology, 2775; 1:400), and anti-SNCA (BD-Biosci, 610,787; 1:250). For the immunofluorescence assays, the sections were then washed and incubated with Alexa Fluor 488-conjugated donkey anti-mouse IgG (Invitrogen, A21202; 1:1000), Alexa Fluor 488-conjugated goat anti-rabbit (Invitrogen, A11008; 1:1000), Alexa Fluor 555-conjugated goat anti-rabbit IgG (Invitrogen, 21,432; 1:1000) or Alexa Fluor 555 goat anti-mouse IgG (Invitrogen, 21,422; 1:1000) for 1 h at room temperature. After a final wash step with PBS, the sections were mounted onto glass slides, and ProLong gold anti-fade reagent containing DAPI (Life Technologies, {"type":"entrez-protein","attrs":{"text":"P36931","term_id":"2506707","term_text":"P36931"}}P36931) was applied for visualizing nuclei. For the IHC, the slides were incubated with streptavidin-HRP (Thermo, SA10001; 1:1000) for 40 min and then stained with DAB (Shanghai Gene Company, GK500705) substrate. Slides were observed and images were taken under a confocal microscope (Axiovert LSM510, Carl Zeiss Co., Germany). The immunostaining signals were quantitatively analyzed using the Optical Fractionator method with Microbrightfield Stereo-Investigator software (Stereo Investigator software; Microbrightfield). The average SNCA and NLRP3 intensities were calculated using ImageJ by sampling a 30 × 30 pixel area, and 36 images were captured from six consecutive mesencephalon sections. The values were reported as the average intensity above the background ± SD. -