Immunohistochemical Analysis of Bone Marrow

Femurs (one each from two mice per group for 8wk HU and both femurs for one mouse per group for HUR) were carefully excised and fixed in 10% neutral buffered formalin, decalcified with Immunocal Decalcifier (Statlab 1414-1), processed (Sakura Tissue-Tek VIP5), embedded in paraffin, sectioned at 4 µm, and mounted on plus slides. The slides were stained with Tyrosine Hydroxylase antibody (EMD Millipore, AB152 1 : 500) or Leptin/OB (Novus Biologicals MAB498, 1 : 4000) and the BOND Polymer Refine DAB Detection kit (Leica DS9800) on a Leica BOND-MAX immunostaining platform, dehydrated in graded alcohols, cleared in xylene, and cover slipped with Cytoseal 60 (Thermo Scientific 8310). For each stain, four sections per femur were analyzed. The BM of each IHC section was randomly photographed at 100 × 3 times in each of three regions: proximal (in or near the femoral head), medial (within the shaft of the bone), and distal (in or near the epicondyles). Micrographs were scored using the IHC Profiler^{108 (link)} plugin for ImageJ, which allows for non-biased, automated scoring of histological slides. In this method, each DAB-stained pixel is categorized into one of four pre-set pixel intensity bins (High Positive, Positive, Low Positive, Negative) and yields the percent contribution of each bin to the image.