Lr white resin

Scanned tibial fractures were processed to plastic, sectioned and stained. Briefly, tibial fractures were dehydrated using a graded series of ethanols and infiltrated and embedded in LR White resin (London Resin Company limited, Reading, England). Samples were polymerised in LR White resin at 60 °C for 24 h. Five micron thick longitudinal sections were cut at the midpoint of undecalcified callus on a Leica RM 2155 Rotary Microtome (Leica, Wetzlar, Germany) with a tungsten carbide blade. Sections were stained using Safranin O and Fast green to examine bone and cartilage content. Additional sections were also stained for the presence of tartrate-resistant acid phosphatase (TRAP; commonly used as a cytochemical marker of osteoclasts). The total area of the callus stained positive for TRAP activity was divided by the total callus area to yield the percentage of the callus occupied by TRAP activity as a surrogate marker for osteoclast area. Sections were photographed on a Leica DMBRE microscope (magnification 25x for Safranin O and Fast green stained sections and 50x for TRAP stained sections) before being assessed both qualitatively and quantitatively using Leica Qwin software.

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).

Following the removal of the right testis, tissues were immediately sliced into small pieces (~2 mm³) and then fixed in paraformaldehyde (4%) in phosphate buffer pH 7.2. After dehydration of tissues in a graded series of alcohols, the samples were treated with a mixture of LR White resin (Electron Microscopy Sciences, PA, USA) and ethanol (2:1) (v:v) for 1 h at room temperature in order to improve infiltration. The samples were then embedded in LR White resin and sectioned at a thickness of 700 nm using a Leica EM UC7 ultramicrotome (Wetzlar, Germany). Semi-thin sections were stained with 1% toluidine blue/borax (pH 8.4) for 2 min and observed under a Leica DM 750 light microscope (Wetzlar, Germany) [27 ].