Richard allan scientific decalcifying solution

The 20mm mid-VCA segment was fixed in 10% buffered formalin (48 hours) and decalcified over a 7 week period in Richard-Allan Scientific™ Decalcifying Solution (Thermofisher, Waltham, MA). Micro-CT scanning was performed using an Inveon PET CT scanner (Siemens Medical Solutions USA, Inc., Malvern, PA) using settings of 80 kV and 500uA and imaging software (PMOD Technologies, Zurich, Switzerland) at a medium-high magnification resolution. We used the bone microarchitecture application of AnalyzePro software (Analyze, Mayo Clinics, Rochester, MN) to measure total transplant volume, cortex volume, and medullary canal volumes. The segmentation portion of the application was used to separate the contrast-filled cortical and medullary vessels from surrounding bone. Capillary density within the allotransplants and normal tibiae were then calculated, reported as a percentage of total bone, medullary space and cortical bone volumes occupied by vessels.

Tibias were placed in Richard-Allan Scientific™ Decalcifying Solution (ThermoFisher Scientific, Waltham, MA) for 2 days, subjected to sequential ethanol dehydration, and embedded in paraffin. Embedded samples were then sectioned (5 μm) on a transverse plane, and sections were stained with hematoxylin and eosin (H&E) for subsequent analysis of static bone parameters [19 (link)]. Osteoblasts were identified as 3 or more cuboidal cells lining the trabecular bone surface, osteoclasts were identified as large, multinucleated cells on the bone surface (often adjacent to eroded surface), and bone marrow adipocytes were identified as white ‘ghost cells’ within the marrow area and excluding vasculature and/ or sectioning artifacts. All analyses were performed using BioQuant® Osteo 2018 version 18.2.6 (BioQuant® Image Analysis Corporation, Nashville, TN). The region of interest in the tibia was a 1 mm long region of the cancellous area starting 200 μm proximal to the distal growth plate.

Animals were sacrificed and femurs isolated for immunohistochemistry and preserved in 4% paraformaldehyde. Samples were decalcified for 12 h using Richard-Allan scientific decalcifying solution (Thermo Scientific) and embedded in paraffin for sectioning. Before immunohistochemical staining antigens were retrieved (sections were microwaved in a 10-mmol/L citrate buffer at pH 6.0), and endogenous peroxidase activity was quenched with peroxidase blocking buffer (Dako). Samples were incubated with 10% normal goat serum to prevent nonspecific binding. Sections were incubated with an anti-hNIS primary antibody (1:50, Abcam). Secondary goat anti-rabbit horseradish peroxidase–conjugated antibody was used (Dako, EnVision + System, HRP) and immunoreactivity was visualized with 3,3′-diaminobenzidine. Cells were counterstained with Mayer hematoxylin, and stainings were analyzed using a Nikon Eclipse 80i microscope (Nikon).