Image pro express

Hearts were harvested immediately after animals were sacrificed by cutting the abdominal aorta under deep anesthesia on day 21. After measuring the weight (mg), midventricular slices of the heart were stained with hematoxylin and eosin and Mallory methods. Blue staining of collagen fibers was quantified as a measure of fibrosis using the Image-Pro Express software program (Media Cybernetics, Rockville, Maryland). The area of the myocardium affected by cell infiltration was determined as infiltrated. All data were analyzed in a blind fashion by 2 independent investigators and averaged.

Graft middle cable of sham, treatment, and control groups was harvested and immediately fixed in 2.5% glutaraldehyde. The grafts were then embedded in paraplast paraffin, cut in 5μm, and then stained with toluidine blue. Morphometric analysis was performed using an image analyzing software (Image-Pro Express, version 6.0.0.319; Media Cybernetics, Silver Springs, MD). Equal opportunity, systematic random sampling, and two-dimensional dissector rules were followed to cope with sampling- related, fiber-location-related, and fiber-size related biases.

Femurs and ulnae were dehydrated in graded alcohols, cleared in xylene, and embedded in MMA following standard protocols.³⁹ (link) For femurs, thick-cut sections were taken ~3 mm proximal to the tibiofibular junction and manually ground down to ~30 μm. For ulnae, thick-cut sections were taken ~2 mm proximal and ~2 mm distal to the ulna midshaft and manually ground down to ~30 μm. A single unstained section from each bone was imaged digitally on a fluorescent microscope using filter sets that provided excitation and emission for the calcein and alizarin wavelengths. Digital images were imported into Image-Pro Express (Media Cybernetics, Inc.), and the following histomorphometric measurements were recorded for the periosteal surface: total perimeter, single label perimeter (sL.Pm), double label area (dL.Ar) and perimeter (dL.Pm), total bone, and marrow area. The following results were calculated: mineral apposition rate (MAR = dL.Ar/dL.Pm/11 d), mineralizing surface (MS/BS = (0.5 × sL.Pm + dL.Pm) / total perimeter × 100), and bone formation rate (BFR/BS = MAR × MS / BS × 3.65). Relative formation parameters were calculated for each mouse by subtracting the nonloaded (left ulna) values form the loaded (right ulna) values. Histology sections, staining, and analyses were conducted by the Histology Core Facilities within the Indiana Center for Musculoskeletal Health.

Following 60 days of treatment, left tibiae were fixed by immersion in buffered formalin for 72 h at room temperature, then decalcified in 10% ethylenediaminetetraacetic acid for 4 weeks, dehydrated in a desiccator with graded ethanol (25, 50, 70, 90 and 100%), defatted in xylene and embedded in paraffin. Longitudinally oriented, 5-mm-thick sections were cut and stained with H&E at room temperature for 10 min for histopathological analysis, and with Safranin-O/Fast green dye for 10 min at room temperature for histomorphometrical analysis. Measurements were taken using a light microscope (magnification, ×4; Leica Microsystems GmbH, Wetzlar, Germany) and an image analyzer (Image Pro-Express; Media Cybernetics Inc., Rockville, MD, USA). Static parameters including bone volume per tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp) were calculated and expressed as previously described (13 (link),14 ).