Epothin

Following μCT scanning, bones were dehydrated in a graded series of ethanol solutions and embedded in a non-infiltrating epoxy resin (EpoThin, Buehler). A series of 700 μm thick transverse sections were made spanning the length of the humeri using a low-speed wafering blade (Phoenix 4000, Buehler). Sections were then fixed to plastic slides and ground and polished to a mirror finish using a series of grit papers followed by decreasing diameters (30 and 9μm) of colloidal diamond suspensions (Metadi, Buehler). Trabecular measurements were made on sections from the proximal humerus, between 1–1.5 mm distal to the proximal growth plate, while cortical measurements were made on diaphyseal sections cut from the cortical midshaft, just distal to the deltoid tuberosity, similar to the region of interest used for μCT analysis.

After μCT scanning, femurs were dehydrated in a graded series of alcohol and embedded in an epoxy resin (EpoThin, Buehler). Roughly 1 mm thick slabs at the distal femoral ROI were cut from the embedded specimens (Isomet 5000, Buehler) and fixed to plastic slides. The slabs were ground to a thickness of roughly 500 μm and polished to a mirror finish (Phoenix 4000, Buehler), using a series of colloidal diamond suspensions (9, 3, 1 μm average particle size, Metadi, Buehler) with ethylene glycol as a lubricant.

After μCT scanning, femurs were dehydrated in a graded series of alcohol solutions and embedded in an epoxy resin (EpoThin, Buehler). 1 mm thick slabs at the femoral ROI were cut from the embedded specimens (Isomet 5000, Buehler) and fixed to plastic slides. The slabs were ground to a thickness of roughly 500 μm and polished to a mirror finish (Phoenix 4000, Buehler), using a series of colloidal diamond suspensions (9, 3, 1 μm average particle size, Metadi, Buehler) with ethylene glycol as a lubricant [14 ].

Humeri from two oim/oim and two +/+ 8-week old female mice were harvested and cleaned of surrounding soft tissue. The bones were left to dry in air for an hour and embedded in epoxy resin (EPOTHIN; Buehler, Lake Bluff, USA). The epoxy resin and hardener were mixed and let cool for 10 minutes prior to embedding the bones, in order to increase its viscosity and avoid the infiltration of resin into the pores. The resin-cast bone samples were left to polymerize at room temperature. The embedded humeri were then cut transversally at the mid-diaphysis using a low speed diamond saw (Isomet, Buehler GmbH, Germany). The proximal sections were cut in cubes and polished using increased grades of carbide papers (P800 to P4000). The sections were further polished using alumina powders (0.3 μm and 0.05 μm) to obtain a mirror-like surface for AFM scan. The finished surface has roughness less than 10 nm RMS when observed from 1×1 μm² images. Samples were stored under ambient conditions (24°C and 60% RH) for further AFM characterizations.