Rabbit anti human il 33 mab

Confluent cell monolayers were harvested using cell lysis buffer^® (Cell Signaling Technology) according to the manufacturer’s instructions. Nuclear and cytoplasmic extracts were separated using NE-PER nuclear and cytoplasmic extraction reagents (Pierce Biotechnology, Rockford, IL), respectively. Whole cell lysates, as well as nuclear and cytoplasmic fractions were separated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes (ATTO, Tokyo, Japan) using a semidry transblot system (Bio-Rad Laboratories, Hercules, CA). Non-specific binding on the blots was blocked with 0.5% (w/v) non-fat dried milk and 0.1% (v/v) Tween 20 in PBS overnight at 4°C, followed by incubation for 1 h at RT with rabbit anti-human IL-33 mAb (EPITOMICS), mouse anti-human GAPDH mAb (MBL, Tokyo, Japan), rabbit anti-human p38 (Cell Signaling Technology), or with p38 polyclonal antibodies (Cell Signaling Technology) at 1:1,000 for 1 h at RT. Blots were incubated with HRP-conjugated goat anti-rabbit IgG (Cell Signaling Technology) at 1:2,000 for 1 h at RT and then soaked in Immunostar^® (Wako Pure Chemical Industries). Chemiluminescent signals were detected using an LAS-4000IR luminescent image analyzer (Fujifilm, Tokyo, Japan). The intensity of expression was quantified by densitometry using ImageJ software.

Gingival tissues from the junctional epithelium of patients with chronic periodontitis (n = 5) were obtained from teeth that required extraction at the time of initial examination. The criteria for chronic periodontitis comprised: probing pocket depth ≥ 5.0 mm, bleeding upon probing, clinical attachment loss ≥ 5.0 mm and radiographically confirmed extensive bone loss. The criteria for periodontally healthy individuals comprised: probing pocket depth < 3.0 mm, no bleeding upon probing, clinical attachment loss < 3.0 mm, and scarcely bone loss confirmed radiographically. Frozen gingival tissues embedded in OCT compound (Sakura Finetek, Tokyo, Japan) were sliced into 4-μm-thick sections. Endogenous peroxidase was blocked by incubation with 0.5% H₂O₂ for 5 min. The sections were then incubated with 2 μg/ml of rabbit anti-human IL-33 mAb (EPITOMICS, Burlingame, CA) or an isotype-matched control for 1 h at RT and with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cell Signaling Technology, Beverly, MA). The sections were immunostained with 3,3’-diaminobenzidine (DAB) substrate (DakoCytomation, Carpinteria, CA), counterstained with Mayer’s hematoxylin and observed by microscopy.