Goat anti mouse fitc

The deparaffinized sections were subjected to heat mediated antigen retrieval, and blocked in H₂O₂ for 30 minutes, followed by PBS with 5% BSA and 0.2% Triton X-100 at room temperature for 30 minutes, and then stained overnight with primary antibody against NLRP3 (Abcam, Cat#ab214185), Prx1 (Origene, Cat#TA803116), USP1 (CST, Cat#D37B4) or UCHL5 (Abcam, Cat#ab133508) at 4 °C. For immunohistochemistry staining, after rinsing with PBS for 15 minutes, tissues were incubated with HRP-conjugated secondary antibody at room temperature. Sections were then washed and colors were developed with DAB (3,3ʹ-diaminobenzidine). Next, hematoxylin was used as a counterstain. For immunofluorescence staining, tissues were incubated with goat anti-mouse FITC (Proteintech, Cat#SA00003-1) or goat anti-rabbit Cy3 (Proteintech, Cat#SA00009-2) at room temperature. Slides were mounted with Mounting Medium containing DAPI (Vector Labs, Burlingame, CA, USA). All the images were captured with a Leica DM4000 fluorescence microscope.

Daphnetin (purity>98%, catalog number:110900) was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Daphnetin was diluted in culture medium with 0.1% dimethylsulfoxide (DMSO, Sigma, St. Louis, MO, USA) and then sterile-filtered before used. All controls and samples contained the same concentrations of DMSO.
Concanavalin A (ConA) and 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The mAbs against mouse CD3, CD4, CD8, IFN-γ, IL-2, IL-4 and IL-6 ELISA kits were purchased from Biolegend (California, USA). RPMI-1640 medium was purchased from HyClone (Logan, UT), and fetal bovine serum (FBS) was obtained from Invitrogen-Gibco (Grand Island, NY). Primary antibodies used for Western analysis Calmodulin, Calcineurin (CaN), Ca²⁺/Calmodulin-dependent kinase II (CaMKII), P-CaMKII, NF-κB, which were purchased from Cell Signaling (beverly, MA), NFAT2 antibodies was purchased from Abcam (Cambridge, MA), β-actin was obtained from Tianjin Sungene Biotech Co., Ltd. Goat anti-mouse IgG, Goat anti-mouse FITC, Peroxidase-conjugated AffiniPure Goat anti-mouse IgG (H + L) were purchased from Protein Tech Group, 2, 4-Dinitrofluorobenzene (DNFB) and cyclophosphamide (CTX) were obtained from Sigma-Aldrich (St. Louis, MO).