Digital imaging hub

After μCT imaging, femurs were decalcified for three days in 20% EDTA at 4°C. Decalcified bones were processed and embedded in paraffin before sectioning at 4μm thickness through the infectious nidus and bone marrow cavity using a Leica RM2255 microtome. Sectioned femurs were stained with a modified hematoxylin and eosin (H&E) that included orange G and phloxine for enhanced bone contrast, tartrate-resistant acid phosphatase (TRAP) stain with hematoxylin counterstain, or 3,3’-diaminobenzidine (DAB) immunohistochemistry to detect myeloperoxidase (MPO). OsteoMeasure software (OsteoMetrics, Inc., Decatur, GA) was used to manually analyze TRAP-stained histologic sections at a region of interest encompassing the trabeculae proximal to the growth plate in the distal femur. Osteoclast number, osteoclast surface, and bone perimeter were calculated and reported per ASBMR standards [42 (link)]. A Leica SCN400 Slide Scanner was used to scan stained femur sections in brightfield at 20X. Images were uploaded to and imaged with the Digital Imaging Hub (Leica Biosystems, Buffalo Grove, IL) and Tissue Image Analysis 2.0 (Tissue IA 2.0) (Leica Microsystems, Buffalo Grove, IL) was used to analyze callus area of infected femurs at 20X.