Atomic Force Microscopy of Articular Cartilage

Knees from 14-week-old WT and ERp57 cKO mice were dissected, embedded in O.C.T. compound mounting medium (00411243, VWR chemicals, Darmstadt, Germany), and frozen in liquid nitrogen. A Leica CM1950 cryostat (Leica Biosystems, Wetzlar, Germany) was used to generate 20 µm-thick frozen tissue sections. Sections were thawed, submerged in PBS buffer, and analyzed with a NanoWizard I AFM (JPK Instruments, Berlin, Germany) in combination with an inverse optical microscope (Axiovert 200, Zeiss, Göttingen, Germany) as described before [23 (link),54 (link),55 (link)]. Briefly, silicon nitride cantilevers (0.1 N/m, MLCT, Bruker) were used to record 625 indentation curves in an area of 3 × 3 µm². For each sample, 9 areas were measured. The sample stiffness was determined by fitting a modified Hertz model to the force-indentation curves using the JPK Data Processing software (V5.0.96, JPK Instruments, Berlin, Germany). Histograms of the Youngs Moduli were generated and a linear combination of two Gaussian functions was fitted to the histograms utilizing Igor Pro software (Version 6.3.7.2, WaveMetrics).