Surgipath decalcifier 2 solution

Hind limb tissue harvested at euthanasia was fixed in 10% neutral buffered formalin for 24 hours, decalcified using Surgipath Decalcifier II solution (Leica Biosystems Inc., Buffalo Grove, IL), embedded in paraffin, and sectioned using 3–5 μm tissue thickness. Hematoxylin and Eosin (H&E) staining of operated mouse paws was performed to document differences in histological architecture of the soft tissues of the paw between treatment and control groups. Thickness of the Malpighian layer, consisting of the distance from the bottom of the stratum basale to the top of the stratum spinosum of the epidermis, was measured with ImageJ using at least 3 images per cross-section taken at 20x magnification using a BZ-X800E microscope (Keyence Corporation of America, Itasca, IL). Picrosirius red staining of operated mouse paws was performed to detect differences in fibrotic response between the two groups. Staining was performed by bathing cross-sections in picrosirius red solution (Sigma-Aldrich, St. Louis, MO) for 40 minutes, followed by dehydration and mounting. Collagen deposition was quantified using at least 3 images per cross-section taken at 20x magnification by measuring percent collagen area (in red) divided by total tissue area within the dermis.