Rabbit anti mouse il 1β antibody

Representative paws from each group in the CIA model were used to examine joint pathology and cytokine expression. Decalcified joint tissue section preparation, Trichrome staining and cytokine detection by immunofluorescence were undertaken as previously described^{11 (link), 12 (link), 40 (link)}. To detect cytokine expression, the joint sections were de-waxed in Xylene, and re-hydrated by incubation with decreasing concentrations of ethanol before incubation in citrate buffer (10 mM citric acid, 0.1% Tween, pH 6.0) for antigen retrieval. Sections were then blocked against endogenous biotin (Molecular Probes, Oregon, USA) and incubated with goat anti-mouse IL-17 antibody (Abcam, Cambridge, UK) or rabbit anti-mouse IL-1β antibody (Abcam) for 12 h at 4 °C. Signal was detected using biotinylated secondary antibodies (anti-goat IgG for IL-17 and anti-rabbit IgG for IL-1β) and Streptavidin-Alexa Fluor647. Sections were then dehydrated by incubation in increasing concentrations of ethanol, mounted and counterstained using VECTORSHIELD mounting medium containing DAPI (Vector Laboroatories, California, USA). Immunofluorescence images were obtained using an LSM 510 META confocal laser coupled to an Axiovert 200 microscope (Zeiss) and analysed using Zeiss LSM Image Browser software (Oberkocken, Germany).

Decalcified joint tissue section (7 μm) preparation, Haematoxylin and Eosin (H & E) and Trichrome staining and detection via immunofluorescence were performed as previously described [7,13] . The extent of synovitis, pannus formation, and destruction of bone and cartilage was determined using a graded scale: grade 0, no signs of inflammation; grade 1, mild inflammation with hyperplasia of the synovial lining and minor cartilage damage; grades 2 through 4, increasing degrees of inflammatory cell infiltrate and destruction of bone and cartilage. To detect IL-1β expression, sections were incubated with sodium citrate buffer (10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0) for antigen retrieval and stained with a rabbit anti-mouse IL-1β antibody (Abcam; rabbit IgG isotype control) with DAPI as a counterstain, at 4 °C for 12 h, followed by detection using a biotinylated goat anti-rabbit IgG antibody and streptavidin–Alexa Fluor 647. Immunofluorescence images were obtained using an LSM 510 META confocal laser coupled to an Axiovert 200 microscope (Zeiss) and analysed by Zeiss LSM Image Browser software.