Anti il 1β antibody

The mice were euthanatized by cervical dislocation under general anesthesia with intraperitoneal injection of mixture of medetomidine (0.3 mg/kg), midazolam (4 mg/kg), and butorphanol (5 mg/kg) on day 2 after LPS application, and on day 7 after alkali burn injury. Enucleated eyes were embedded (Tissue-Tek OCT Compound; Sakura Finetek Japan, Tokyo, Japan), and a 7-μm-thick section was made using a cryostat (HM505; Microm, Walldorf, Germany). These thin sections were stained by anti-Gr-1 antibody (marker of neutrophils, dilution 1:100, BD Biosciences, Franklin Lakes, NJ), anti-IL-1β antibody (dilution 1:100, Abcam, Cambridge, United Kingdom), and anti-MMP-9 antibody (dilution 1:250, Abcam) as a first antibody overnight in 4 °C, respectively. They were then stained by the indirect enzyme-antibody method (avidinebiotin complex method; Histofine®, Nichirei Biosciences, Tokyo, Japan). The section, obtained from enucleated cornea, was examined by light microscopy (BH-2, Olympus, Tokyo, Japan). Each section was observed at a working magnification of X 200 (X 20 objective lens and X 10 ocular lens; 0.723 mm2 field of view). Central cornea stroma in each mouse was observed and the cells with positive brown staining in the corneal stroma were counted.

For IHC staining, the procedures were as described previously [23 (link)]. After deparaffinating and rehydrating, the 5 μm thick tumor tissue sections were heated by water bath with citric acid buffer for 20 min and blocked in 5% normal goat serum for 30 min, and then incubated with either anti-NLRP3 antibody (1:400, sigma, St Louis, MO, USA) or anti-IL-1β antibody (1:200, Abcam, Cambridge, UK) at 4 °C overnight. Subsequently, the GTVision Two-step Visualization System (Genetech, Shanghai, China) was used to visualize the immunostaining and hematoxylin was used to counter-stain. Finally, the sections were examined under light microscopy and evaluated by the Immuno-Reactive-Score (IRS), which was calculated by multiplying staining proportion score (PS) and staining intensity score (IS). PS was defined as 0 (0%), 1 (1%~ 25%), 2 (26%~ 50%), 3 (51%~ 75%), and 4 (76%~ 100%). IS was classified as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). According to the obtained IRS scores, patients were divided into three groups: negative expression group with IRS = 0, weak expression group with IRS = 1~ 3 and strong positive expression group with IRS = 4~ 12.

Mouse islet β-TC-6 cells were purchased from TONGPAI (Shanghai) Biotechnology Co. Ltd. PCB118 was purchased from AccuStandard Inc. (New Haven, CT, USA). Cell Counting Kit-8 (CCK8), ROS detection kit, and DMSO were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Specific antibodies against NLRP3, caspase-1, and fetal bovine serum were purchased from Cell Signaling Technology (CST, Boston, MA, USA). Anti-caspase-1 antibody (EPR4321) and anti-IL-1 β antibody were purchased from Abcam (USA). GAPDH polyclonal antibody was purchased from BioWorld (USA). ELISA kits for IL-6, IL-18, and CCL-2 were purchased from Cheng Lin Biological (Beijing, China).

Twenty-six days after LPS induction, IL-1β, IL-6, TNF-α, cyclooxygenase-2 (COX-2), and NF-κB concentrations in the hippocampus were assayed according to a previously described procedure (Lee et al. 2014 (link)). Three rats from each group were deeply anesthetized via isoflurane inhalation (1.2%), and were sacrificed one day after behavioral testing. The IL-1β, IL-6, TNF-α, COX-2, and NF-κB concentrations were evaluated with competitive enzyme-linked immunoassays (ELISAs) using anti-IL-1β antibody (Abcam, Cambridge, MA, USA), anti-IL-6 antibody (Abcam), anti-TNF-α antibody (Abcam), anti-COX-2 antibody (Abcam), and anti-NF-κB antibody (Abcam) according to the manufacturer's protocols.

After fixation, 5-μm sections were processed for immunohistochemical analysis with anti-NLRP3 antibody (1:500, Abcam, Cambridge, MA, United States), anti-ASC antibody (1:500, Abcam, Cambridge, MA, United States) and anti-IL-1β antibody (1:500, Abcam, Cambridge, MA, United States). The negative control used serum (Boster Biological Technology, Wuhan, China) instead of primary antibody and the immunoreactivity was visualized by the Elite ABC kit (BioGenex, San Ramon, CA, United States). Three independent observers were asked to assess the intensity of immunostaining (Shi et al., 2004 (link); Kim et al., 2005 (link)) and the evaluation of relative staining levels was repeated at least four times (Zhang W. et al., 2011 (link)).

Briefly, protein in liver tissues was extracted using RIPA lysis buffer at 14,000 rpm for 15 min at 4°C. Protein concentration was determined using bicinchoninic acid (BCA). Protein was loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes and blocked with 8% skim milk. Then, membranes were incubated with 1 : 1000 dilution of rabbit anti-Nrf2 antibody (CST, USA), anti-HO-1 antibody (San Ying Biotechnology, China), anti-ASC antibody (CST, USA), anti-TXNIP antibody (San Ying Biotechnology, China), anti-NLRP3 antibody (Beyotime Biotechnology, China), anti-ASC antibody (San Ying Biotechnology, China), anti-caspase-1 antibody (Abcam, USA), and anti-IL-1β antibody (Abcam, USA) and 1 : 10,000 dilution of mouse anti-β-actin antibody (San Ying Biotechnology, China) overnight at 4°C. After washing with PBST, the blots were incubated with 1 : 10,000 dilution of secondary antibodies (Abmart, China). Finally, the proteins were observed and photographed with the ECL (electrochemiluminescence) system. The protein electrophoresis bands were analyzed by the ImageJ software normalized to β-actin as a reference.