The pheno type was assessed at the time of experiments by immu nolabelling for collagen type II and aggrecan, using the primary antibodies polyclonal rabbit anti human col lagen II and monoclonal mouse anti human aggrecan. The secondary antibodies used were polyclonal goat anti rabbit IgG conjugated with Alexa Fluor 594 and polyclonal rabbit anti mouse IgG conjugated with Alexa Fluor 488. Identification of ChemR23 and chemerin was performed with the pri mary antibodies polyclonal rabbit anti human ChemR23 antibody, and polyclonal goat anti human TIG 2 antibody. The secondary antibodies used were goat anti rabbit IgG conjugated with Alexa Fluor 488, and anti goat IgG conjugated with Alexa Fluor 594. Chondrocyte cultures were grown on fibronectin coated chamber slides for 24 h, and for seven days.
The cultures were washed twice with phosphate buffered saline dig this and fixed for 10 minutes in cold PBS containing 0. 2 M sucrose and 4% paraformaldehyde. After fixation, the slides were blocked for one hour with PBS containing 1% bovine serum albumin. Thereafter, cell cul tures were incubated at 4 C overnight with the primary antibodies. The slides were then washed three times in PBS and incubated with secondary antibodies for one hour in room temperature. Isotype control was used to assess non specific binding. The slides were mounted by adding DAPI fluoromount G and examined in a Zeiss axiophot photomicroscope. Immunohistochemistry Immunohistochemical studies were performed to inves tigate whether ChemR23 and chemerin were present in native cartilage tissue.
Biopsies were fixed in paraformal dehyde containing 0. 2 M sucrose discover this in PBS. After 48 h, the tissue was embedded in paraffin and sectioned at 5 um thickness onto poly L Lysine coated slides. Sections were deparaffinised by xylene and graded alcohol washes and immersed in dis tilled water. Thereafter, sections were incubated in PBS containing 1% BSA for 60 minutes followed by incuba tion with monoclonal mouse anti human ChemR23, diluted at 1100 and incubated at 4 C overnight. After rinsing in PBS, sections were incubated for 45 minutes with secondary goat anti mouse antibody conjugated with horseradish peroxidase. For the detection of chemerin, polyclonal goat anti human TIG 2 was used, followed by an Alexa Fluor 594 conjugated donkey anti goat IgG antibody for detection. Sections were mounted by adding DAPI fluoromount G.
Matched isotype antibodies were used as a control for non specific back ground staining. Western blotting Intracellular signal transduction in chondrocytes stimu lated with chemerin was investigated by immunoblotting of phosphorylated MAPKs p4442 and phosphorylated Akt. To detect the phosphory lated MAPKs, a phospho Erk12 pathway sampler kit was used. Phospho specific antibody towards phospho Akt was used to detect the ChemR23 mediated phosphorylation of Akt.