Prior to histological processing, the scanned right femora of the F1 dams were fixed in 4% paraformaldehyde and 0.1 M sodium cacodylate fixative for 48 h, followed by 3 washes for 30 min, and then stored in a solution containing 0.1 M sodium cacodylate and 10% sucrose [42 (link)]. The femora were processed to plastic, then sectioned and stained. Briefly, the right femora were dehydrated using graded concentrations of ethanol (70%, 90%, and 100%), and the samples were infiltrated and embedded in LR White resin (London Resin Company limited, Reading, England) and polymerized in LR White resin at 60 °C for 24 h. Longitudinal plastic sections were cut at 5 µm, at the midpoint of the undecalcified femora using a tungsten carbide blade on a Leica RM 2155 Rotary Microtome (Leica, Wetzlar, Germany). Sections were stained with Light Green and then counter-stained with Safranin O to examine bone and cartilage content [42 (link)]. Stained sections were photographed at 25× magnification using Leica IM50 imaging software (Heerbrugg, Switzerland), and then viewed using a Leica DFC420 Light Microscope (Heerbrugg, Switzerland). The following parameters were measured: growth plate thickness (µm), trabecular bone area (percentage), and calcified cartilage (percentage), using the Leica Qwin V3 Standard software (Heerbrugg, Switzerland).
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