Zb 2307

The tissue sections were obtained from the Department of Pathology of the First Affiliated Hospital of Guangxi Medical University. IHC assay was performed using a universal two-step IHC kit (PV-9000, ZSGB-BIO, Biotech, Beijing, China) following the manufacturer’s protocols. The primary antibodies against CCR1 (DF2710, Affinity, Jiangsu, China), CCR5 (AF6339, Affinity, Jiangsu, China), and CCR7 (AF5293, Affinity, Jiangsu, China), as well as peroxidase-conjugated goat antirat IgG (ZB-2307, ZSGB-BIO, Beijing, China) were used to perform the IHC assay. Tumor sections were incubated overnight with primary antibodies at 4° C. The primary antibody titer was configured according to the IHC concentration recommended by the manufacturer (CCR1, 1:200; CCR5, 1:300; CCR7, 1:100).

J774A.1 cells, a mouse monocyte/macrophage cell line, were propagated in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO₂.
T. pyogenes (strain 0912) was cultured in Martin broth medium with 10% fetal bovine serum under aerobic conditions.
Primary antibodies included rabbit monoclonal anti-GSDMD (ab209845, Abcam), rat monoclonal anti-caspase-1(BL-645102, Biolegend), rabbit monoclonal anti-caspase-11 (ab180673, Abcam), mouse monoclonal anti-NLRP3(AG-20B-0014, Adipogen), and mouse anti-GAPDH antibody (GTX627408, GeneTex). Secondary antibodies included horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (ZB-2305, ZSGB-BIO), HRP-conjugated goat anti-rabbit IgG (ZB-2301, ZSGB-BIO) and HRP-conjugated goat anti-rat IgG secondary antibody (ZB-2307, ZSGB-BIO).
Chemicals included VX-765 (Capase-1 inhibitor) (S2228, Selleck), MCC950 (NLRP3 inhibitor) (S8930, Selleck), Necrostatin-1 (Nec-1) (receptor-interacting serine/threonine protein kinase 1 [RIP1] inhibitor) (A4213, APExBIO), Necrosulfonamide (NSA) (mixed lineage kinase domain-like protein [MLKL] and GSDMD inhibitor) (B7731, APExBIO), and Wedelolactone (Wed) (caspase-11 and NLRP3 inhibitor) (HY-N0551, MedChemExpress).

Immunohistochemical staining to determine levels of DAT and D₂R proteins and TH activity was conducted with striatal slices. To prepare for staining, the slices were prewashed in PBS (0.01 M, pH 7.4) three times for 5 min each, then sequentially treated with 0.2% Triton X-100 in PBS for 5 min and with 0.3% H₂O₂ in PBS for 10 min, and washed in PBS three times for 5 min each, all at room temperature. Slices were initially incubated with 10% normal goat serum for 10 min (or normal donkey serum for TH measurement), then incubated for 20 h at 4°C with the primary antibodies (D₂R antibody AB5084P, Millipore, CA, USA, 1:200 dilution; DAT antibody MAB369, Millipore, CA, USA, 1:100 dilution; TH antibody T1299, Sigma, CA, USA, 1:10,000 dilution), washed three times in PBS, and incubated for 60 min at 37°C with the secondary antibodies (anti-rabbit antibody PV-6001, ZSGB-BIO, CA, USA; anti-rat antibody ZB-2307, ZSGB-BIO, CA, USA; anti-mouse antibody PK4002, Vector, CA, USA). Slices were then washed five times for 3 min each in PBS, and incubated in 100 μL of 3,3′-diaminobenzidine tertrahydrochloride (DAB) for 3 min. The slices immunostained for TH were incubated in the ABC (VECTASTAIN ABC kit, ZSGB-BIO, Beijing, China) reagent for 30 min at 37°C and washed five times for 3 min each in PBS prior to the DAB treatment. Final wash of the slices was done in distilled water.