2030 rotary microtome

Fixed bones were processed on an automated Thermo Electron Excesior tissue processor for dehydration, clearing, and infiltration using a routine overnight processing schedule. Samples were then embedded in Surgipath-embedding paraffin on a Sakura Tissue Tek II-embedding center. Paraffin blocks were sectioned at 5 µm on a Reichert Jung 2030 rotary microtome and were H&E stained. Femur sections were examined by microscopy at 4x optical zoom and digital images obtained. Images were examined blind to the section’s condition. The marrow area starting at 170 µm from the growth plate and extending 2000 µm toward the diaphysis was measured, by outlining the region and quantifying the area using ImagePro software. Adipocytes greater than 30 µm in diameter were counted and expressed relative to the total marrow area. Analyses were done blinded to conditions.

Femurs were fixed in 10% formalin and transferred to 70% ethanol after 24 hours. Fixed samples were processed on an automated Thermo Electron Excesior tissue processor for dehydration, clearing, and infiltration using a routine overnight processing schedule. Samples were then embedded in Surgipath-embedding paraffin on a Sakura Tissue Tek II-embedding center. Paraffin bocks were sectioned at 5 μm on a Reichert Jung 2030 rotary microtome. Slides were stained for TRAP activity and counterstained with hematoxylin according to manufacturer protocol (387A-1KT, Sigma, St. Louis, MO). Osteoblast and osteoclast surface area was measured and expressed as a percentage of total bone surface in the femur trabecular region ranging from the growth plate to 2 mm distal.
For dynamic histomorphometric measures of bone formation, mice were injected intraperitoneally with 200μl of 10mg/ml calcein (Sigma, St. Louis, MO, USA) dissolved in sterile saline at 7 and 2 days prior to harvest. L3-L4 vertebrae were fixed in formalin at time of harvest then transferred to 70% ethanol 48 hours later. Vertebrae were then embedded, sectioned and examined under UV light. Five images were taken and the distance between the calcein lines (bone formation rate, BFR) and their length along the bone surface was measured and used to calculate mineral apposition rate (MAR).