Anti il 1β ab

For immunofluorescence staining, paraffin-embedded brain sections were prepared as described in our previous studies11 (link). The primary antibodies used in the experiments included anti-HMGB1 Ab (R&D Systems Inc., Minneapolis, MN), anti-AQP4 Ab (Abcam Plc, Cambridge, UK), anti-IL-1β Ab (R&D Systems Inc.), anti-microtubule-associated protein 2 (MAP2) Ab (Abcam Plc), anti-glial fibrillary acid protein (GFAP) Ab (Abcam Plc), anti-ionized calcium-binding adaptor molecule 1 (Iba1) Ab (Wako, Osaka, Japan) and anti-myeloperoxidase (MPO) Ab (Abcam Plc). In order to investigate the cellular source as well as the localization of IL-1β, double immunohistochemical staining was carried out with cell marker antibodies including MAP2, GFAP, iba1 or MPO antibodies and an antibody against IL-1β. The sections were then incubated with secondary Abs conjugated with Alexa-488, Alexa-555 or Alex594, which were purchased from Invitrogen (Tokyo, Japan). Finally, the sections were mounted using VECTASHIELD Hard Set Mounting Medium with DAPI (Vector Laboratories Inc., Burlingame, CA) and observed under an LSM 780 confocal microscopic system (Carl Zeiss Inc., Jena, Germany). The counting was performed in a blinded manner.

Cells were prepared in 12-well plates and infected with PmCQ2 as described above. After infection, supernatants were collected and concentrated using 20% (w/v) trichloroacetate, and the cells were lysed with 1 × SDS Loading buffer (Beyotime, China). Cell lysates were subjected to 12% SDS-PAGE and subsequently transferred onto a polyvinylidene difluoride (PVDF) membrane by electroblotting. The membranes were blocked with 5% non-fat dry milk and then immunoblotted with indicated antibodies (Abs) including anti-IL-1β Ab (R&D, United States), anti-Caspase1-p20 Ab (AdipoGen, United States), anti-ASC Ab (Cell signaling technology, Danvers, MA, United States), anti-Nek7 Ab (Abcam, Cambridge, UK), anti-NLRP3 Ab (Wanlei Life Sciences, Shenyang, China) and anti-GAPDH Ab (Beyotime, Beijing, China). Finally, the distinct protein bands were detected by ECL detection reagent (Biosharp, China).