Ab140039

Immunostaining was performed as previously described.34 (link) Briefly, paraffin sections (4 μm) of the brain were blocked with a solution containing 5% bovine serum albumin (BSA) and 0.3%Triton-X at room temperature for 1 h. After blocking, the sections were incubated with the corresponding primary antibody at 4°C overnight according to the experimental purpose. The following primary antibodies were used: Dectin-1 (1:100, ab140039, Abcam), Iba-1 (1:300, GTX632426, GnenTex), GFAP (1:300, G3893, Sigma), NeuN (1:100, 66836-1-Ig, Proteintech), and SYK (1:50, sc-1240, Santa Cruz Biotechnology). Sections were rewarmed at room temperature for 30 min, then washed with phosphate-buffered saline (PBS) three times, and incubated with secondary antibody at 37°C for 1 h. Then, sections were washed with PBS three times, stained with DAPI for 15 min, and then washed with PBS. Finally, the brain sections were imaged using a fluorescence microscope (Nikon, Japan). The field of interest is the striatum around the hematoma. Three images were taken of each animal at a camera setting of 1206×1208 pixels.

The Western blot was conducted as previously described.7 (link) Briefly, protein samples were obtained from the perihematomal area and BV2 cells using RIPA lysis buffer. Then, protein lysate was centrifuged for 20 min at 12,000 g at 4°C. After centrifuging, the supernatant protein solution was collected. After loading an equal amount of protein onto the gel, the electrophoresis procedure was initiated. The proteins were then transferred to PVDF membranes and blocked with a solution containing 5% bovine serum albumin (BSA) for 1 h at room temperature. After blocking, membranes were incubated with primary antibodies at 4°C overnight. The following primary antibodies were used: Dectin-1 (1:1000; ab140039, Abcam), NLRP3 (1:1000; ab263899, Abcam), ASC (1:2000; sc-33958, Santa Cruz Biotechnology), phospho-SYK (1:1000; AF3315, Affinity Biosciences), IL-18 (1:1000; 60070-1-Ig, Proteintech), GSDMD-N (1:1000; AF4012, Affinity Biosciences), caspase-1 (1:1000; 22915-1-AP, Proteintech), β-actin (1:1000; AF5003, Beyotime Biotechnology), and IL-1β (1:1000; 26048-1-AP, Proteintech). After washing three times with TBST, the membranes were incubated with secondary antibody at room temperature for 1 h and then washed a further three times. Finally, a Bio-Rad Molecular Imager was used to detect protein signals, which were quantified by Image J.