Anti nlrp6

The mice were deeply anesthetized and perfused with 0.9% saline, followed by 4% paraformaldehyde. Whole brains were paraffin-embedded and sliced into 4 μm thick tissue sections for immunohistochemical observation. The brain tissue sections were dewaxed with xylene, hydrated in graded ethanol, incubated with 3% hydrogen peroxide, and incubated overnight at 4°C with the primary antibody anti-NLRP6 (1 : 100; Santa Cruz, USA) and anti-goat secondary antibody (Proteintech, USA). Afterwards, the sections were added with droplets of DAB for incubation and counterstained with hematoxylin. All stained slides were viewed under a microscope (Olympus, Japan).

BV2 microglia were washed with phosphate buffer saline solution; fixed with 4% buffered paraformaldehyde; incubated overnight at 4°C with primary antibodies, including anti-NLRP6 (1 : 500; Santa Cruz, USA) and anti-Iba-1(1 : 500; Novus, USA); and incubated with anti-goat secondary antibody (Proteintech, USA) and anti-rabbit secondary antibody (Proteintech, USA). Then, the cell nuclei were stained with DAPI (Beyotime, China). Cells were observed under an inverted fluorescence microscope (Olympus, Japan).

Treated cells and brain tissue around the hematoma were harvested for western blot. RIPA lysis buffer and PMSF were applied to homogenize the collected sample. A BCA Protein Assay Kit (Beyotime, China) was used to measure the protein concentration. The operation steps of the western blot were the same as those previously described [11 (link)]. The membranes were incubated with anti-NLRP6 (1 : 1,000; Santa Cruz, USA) and anti-GAPDH (1 : 1,000; Abcam, UK) overnight. The bands were visualized using a Gel Doc® XR+ System (Bio-Rad, USA), and the density of the visualized bands were analyzed using ImageJ 1.50b (National Institutes of Health, USA).