Quantifying NET Formation in Brain Tissue

Brain tissues were harvested after perfusion from WT and NLRP3 KO mice in each PBS and LPS-injected group and ground using a strainer. The total proteins were obtained using PRO-PREP protein extraction solution (iNtRON Biotechnology, Seongnam, Korea) and analyzed following the previously used method (6 (link)). In brief, primary Abs, CitH3 (ab5103; Abcam), NLRP3 (# AG-20B-0014; AdipoGen, San Diego, CA, USA), and β-actin (#8457; Cell Signaling Technology, Boston, MA, USA), were used to detect the NET formation in the brain tissue. After overnight incubation with primary Abs, the membrane was incubated with a secondary Ab for 1 h at 25°C. The membrane was developed using Clarity Max Western ECL substrate (Bio-Rad, Hercules, CA, USA) and the ImageQuant LAS 4000 mini (Fujifilm, Tokyo, Japan). Each time, the membrane was rinsed thrice with TBS-T. The signals were quantified using Image J (36 (link)) software.