Trem2

Multiplex staining was performed using a multiplex fluorescent immunohistochemistry staining kit (Yuanxibio) according to the manufacturer’s instructions with the following primary antibodies/fluorescent dyes: TREM2 (#91068, 1:1000, Cell Signaling Technology)/Neon-TSA620, FoxP3 (MAB8214, 1:200, R&D Systems)/Neon-TSA520, and CD163 (ab182422, 1:300, Abcam)/Neon-TSA670. Primary antibodies were sequentially applied, followed by horseradish peroxidase-conjugated secondary antibody incubation (DS9800, Lecia Biosystems) and tyramide signal amplification. Sections were then counterstained with DAPI and scanned using the Pannoramic MIDI imaging system (3D HISTECH).

The brain tissues of mice were collected and lysed with RIPA solution. After electrophoresis, the samples were transferred to PDVF membrane, sealed with 5% skim milk for 1 h, and reacted with primary antibody (NLRP3, 1:1000, Casp8, 1:1000, Gsdmd, 1:1000, Trem2, 1:1000, Cleaved caspase-1, 1:1000, GAPDH, 1:1000, Cell Signaling Technology, USA) at 4 °C overnight. After cleaning, the samples were reacted with goat anti-mice IgG (1:3000; Cell Signaling Technology, Danvers, MA, USA) at room temperature for 1 h.

For western blotting of α-synuclein, mice were killed 24 h after the last MPTP-subacute administration. The ventral midbrain (VM) was excised and homogenized in RIPA buffer (Thermo Fisher) supplemented with protease inhibitor (Thermo Fisher) using a TissueRuptor (Qiagen, Germantown, MD). Protein concentration was determined using a BCA Protein Assay Kit (Thermo Fisher) according to the manufacturer’s instructions. Samples were diluted to 2 µg/µl in the same extraction buffer. For TREM2, mice were killed at day 4 after MPTP-acute administration. The striatum (St) was excised, and proteins were extracted and quantified as previously described.
30 μg of protein per sample were resolved in a Mini-PROTEAN TGX 4–20% gradient gel and transferred to a 0.1 μm nitrocellulose membrane (GE Healthcare, Chicago, IL, USA). Membranes were incubated overnight with primary antibody for mouse a-synuclein (1:1000, Cell Signaling) or TREM2 (1:1000, Cell Signaling). 4–5 mice per group were used.