Asylum research mfp 3d atomic force microscope

Mechanical measurements were obtained using an Asylum Research MFP-3D atomic force microscope (Oxford Instruments). Fresh tissue samples from DD patients were dissected to obtain an approximately 10 µm cube from the centre of the nodule. This was then embedded in OCT and snap frozen in liquid nitrogen. Using a cryostat, 30 μm longitudinal slices were cut and mounted onto a glass slide. Tissue stiffness was measured through micro-indentation using a 0.072 Nm⁻¹ probe with spherical 5 μm SiO₂ tip (NovaScan) and the cantilever spring constant was calibrated using the thermal fluctuation method. 100 nano-indentation measurements were taken from 10×10 μm squares, with at least three squares per nodule, with the sample in double-distilled water at room temperature. Elastic modulus was calculated using the Hertz model from indentation profiles using MFP-3D software (Oxford Instruments and Igor).

We purchased 1-undecanethiol from Sigma-Aldrich (St. Louis, MO, USA). 3 aminopropyltri-ethoxysilane (APTES), glutaraldehyde (25% aq. solution), Phosphate-buffered saline (PBS pH 7.4) (11.9 mM phosphates, 137 mM sodium chloride and 2.7 mM potassium chloride) and ethanol (200 proof or 100% ethanol) were purchased from Fisher Scientific (Waltham, MA USA). Mica was purchased from Ted Pella (Redding, CA, USA). AC240TS cantilevers (Olympus, Center Valley, PA, USA) were used for non-contact mode imaging in air, while gold coated TR800PB cantilevers (Olympus) were used for force measurements. To prevent contamination, cantilevers were cleaned using an UV/Ozone Procleaner (BioForce Nanosciences Inc. Ames, IA, USA) before use. Imaging and force spectroscopy experiments were performed on an Asylum Research MFP-3D atomic force microscope (Oxford Instruments, Santa Barbara, CA, USA).