Goat anti mouse il 33

Mice were anesthetized and transcardially perfused, first with ice-cold PBS and then with 4% paraformaldehyde (PFA) in PBS. The brains were removed and postfixed in the same fixative for 24 h, then incubated in 10% and 30% (w/v) sucrose in PBS. Free-floating coronal sections (40-µm thick) were prepared with a Cryostat (CM 3050S, Leica). For immunohistochemistry, the following primary antibodies were used: goat anti-mouse IL-33 (R&D Systems, RRID: AB_884269) at 1:200 dilution, mouse anti-NeuN (Merck Millipore, RRID: AB_2298772) at 1:500 dilution, rabbit anti-Iba1 (Wako Chemicals, RRID: AB_839504) at 1:400 dilution, rabbit anti-S100β (Abcam, RRID: AB_882426) at 1:400 dilution, goat anti-c-Fos (Santa Cruz Biotechnology, RRID: AB_2106783) at 1:500 dilution, and rabbit anti-c-Fos (Cell Signaling, RRID: AB_22472111) at 1:200 dilution. Fluorescently labeled secondary antibodies Alexa Fluor 488, 546, 633 at 1:400 dilution (Thermo Fisher Scientific) and DAPI (at 1:50,000 dilution, Roche) were used for signal detection. Images were acquired using Zeiss LSM510 and LSM700 confocal microscopes with Zen software (Carl Zeiss). Images were taken at 20× and 40× magnification. Areas used for quantification were indicated with red boxes. For c-Fos quantification, maximum projection images of serial z-stack sections (21 sections at 0.75 µm) were constructed using Zen software.

R&D Systems Duoset kits were used to measure human and murine IL-33 by ELISA, while western blotting was carried out using polyclonal goat anti-mouse IL-33, goat anti-human IL-33 or goat anti-mouse IL-1α (R&D Systems) with a rabbit anti-goat IgG HRP secondary antibody (Thermo Fisher), and detected using WesternSure Premium reagent (Licor).

Frontal cortex tissues were sectioned and lysed in RIPA buffer containing 0.1% SDS, a protease inhibitor cocktail (Roche), and a phosphatase inhibitor (Sigma Aldrich). Tissue samples were prepared on NuPAGE Bis-Tris Mini Gel (Life Technologies), followed by the transfer to PVDF membrane (Millipore) following a standard protocol. After blocking in 5% skim milk/PBS/0.1% Tween® 20 (PBS-T) for 1 h, the membrane was incubated with the primary antibody overnight at 4°C and then incubated with the secondary andibody for 1 h at room temperature. Gel images were captured by ImageQuant LAS 4000 LAS 4000 mini (GE Healthcare) and analyzed by ImageJ (Schneider et al., 2012 (link)). The following primary antibodies were used: goat anti-mouse IL-33 (R&D Systems, RRID: AB_884269), diluted 1:1000, and mouse anti-β-actin, diluted 1:5000 (Santa Cruz Biotechnology, RRID: AB_626632).