Binding sites for ASF in human OA cartilage and chondrocytes. (a, b) Reactivity for ASF in OA cartilage. (a) Intense reactivity of ASF with chondrocytes and matrix of MS ≥9 cartilage. The inset shows stained complex chondrons. (b) In MS ≤4 regions, positive chondrocytes were scattered across the superficial zone of cartilage (inset and arrows). Bars in insets of (a) and (b): 50 μm. (c) Shown is a quantitative comparison between MS ≤4 and MS ≥9 regions of OA cartilage. Each bar represents the mean percentage of stained (reactive for ASF) chondrons in the respective areas (n = 7; see Additional file 3: Table S3). The asterisk indicates a significant difference of cell reactivity between cartilage areas of mild and severe degeneration (P = 0.0015; n = 7; paired t-test). (d) Subcellular distribution of ASF reactivity in cultured OA chondrocytes. Cells were incubated with biotinylated ASF and stained with streptavidin-phycoerythrin. ASF reactivity (red) was observed using laser scanning microscopy. Nuclei were counterstained with DAPI (blue). ASF, asialofetuin; DAPI, 4′,6-diamidino-2-phenylindole; MS, Mankin score; OA, osteoarthritis; PE, phycoerythrin.