Il 33 antibody

A piece of fast-frozen mouse liver was homogenized in RIPA buffer by Ultra-Turrax® and left on the ice during 40 min, vortexed regularly before a centrifugation of 10000g at 4°C. The supernatant was then recovered before Bradford dosage. 50 μg of total protein extract was then loaded on polyacrylamide gel, transferred to a nitrocellulose membrane before incubation overnight in IL-33 antibody (goat Ab, R&D) in TBS 5% milk and 1 hour in secondary HRP-conjugated rabbit anti-goat antibody (Dako, USA), and followed by a revelation with ECL (Pierce).

2 mm mouse skin taken from the margin of murine wounds or cells stimulated as described was lysed by using RIPA buffer (pH 7.4) containing protease inhibitor cocktail (Roche). To concentrate IL-33 from cell culture medium, mouse IL-33 antibody (R&D) immunocomplexed to Protein A/G agarose (Abmart) was used to capture IL-33. 10 µg of total protein was used for Western blot. IL-33, iNOS, p38 MAPK, AKT and β-catenin were detected by immunoblot with IL-33 antibody (R&D), iNOS antibody (Abcam), p38 MAPK antibody (Cell Signaling), AKT antibody (Cell Signaling) and β-catenin antibody (Cell signaling), respectively.
5 µm of formalin-fixed, paraffin-embedded tissue sections was mounted on glass slides and used for immunohistochemistry and staining. 4% PFA was used to fixate the samples. After 10-min fixation and subsequent pretreated with antigen retrieval solution, the sections were stained with IL-33 (R&D) antibody or iNOS (Abcam) antibody and F4/80 antibody (Santa Cruz) or Ly6G/Gr antibody (Abcam). The sections were reprobed with anti-Goat IgG FITC (Jackson immunoresearch) or anti-Rat IgG TRITC conjugate antibody (KPL), and then mounted in ProLong Gold antifade reagent with DAPI (Invitrogen) and visualized them by the microscope (Leica).

Adipose tissues were fixed in 1% PFA overnight at 4°C. For immunohistology, fixed tissues were proceeded into paraffin tissue sections, then subsequently stained with Hematoxylin and Eosin (H&E) staining. For whole-mount tissue staining, fixed tissues were incised into around 0.5 cm³. Tissues were stained with Caveolin and CD4 overnight at 4°C, and followed by secondary antibodies for 2 h at RT. For immunofluorescence (IF) staining in sorted VAT DC, 100 µL suspension of sorted cells (~20,000 cells) were attached to glass slides with Cytospin at 2000 rpm 7 min. Slides were then fixed with 4% PFA for 10 min at RT, washed in PBS, and dried overnight at RT. After cell permeabilization, slides were incubated with IL-33 antibody (R&D system) overnight at 4°C. Images were taken with Leica DMi8 or Confocal microscopy (Zeiss LSM 710).