Decalcifying solution

Femoral bone tissues were decalcified in decalcifying solution (Sigma-Aldrich Chemicals) and dehydrated in a graded series of ethanol solutions for 18 h. For the histological staining of picrosirius red, femoral bone tissues were then embedded in paraffin and cut into 7 μm sections in thickness. picrosirius red staining was commercially used for detecting bone collagen. The sections were placed on glass slides, deparaffinated, and hydrated with xylene and graded alcohol. These sections were incubated with picrosirius red (Sigma-Aldrich Chemicals) solution overnight at RT. After each slide was mounted in VectaMount Mounting Medium (Vector Laboratories, Burlingame, CA), images were taken using an Axiomager optical microscope system (Zeiss, Oberkochen, Germany).

Mouse legs were fixed in 4% paraformaldehyde solution and incubated in decalcifying solution (Sigma, St. Louis, MO, USA) for 3 days. The legs were then embedded in paraffin and sliced into 3-μm sections for staining. The sections were deparaffinized with o-xylene for 30 min and then rehydrated with 100%, 80%, 70% and 60% (10 min for each step). After rinsing with tap water, nuclei were stained with iron hematoxylin solution for 5 min and then washed with distilled water. Biebrich scarlet-acid fuchsin solution (Sigma) was used to visualize muscle fibers by staining tissue for 10 min. Collagen was then differentiated by staining with 5% phosphomolybdic-phosphotungstic acid solution, after which the sections were rinsed with distilled water. Collagen fibers were stained with 2.5% aniline blue solution for 10 min. After a final wash with distilled water, the slides were dehydrated with 95% ethyl alcohol and cleared in o-xylene. Stained tissue slides were imaged on a Pannoramic MIDI slide scanner (3DHISTECH Ltd, Budapest, Hungary). Manual shots were operated, magnified (40x), and observed under polarized light. Images were analyzed using ImageJ software (NIH, Bethesda, MD, USA; http://rsbwed.nih.gov/ij/). The total blue area was determined as the pixel percentage of the image. Dermal thickness was measured by Pannoramic Viewer version 1.15.2 (3DHISTECH Ltd).

The harvested samples were fixed in 10% neutral buffered formalin (NBF, Leica Biosystem) at room temperature for 24 h. Afterward, decalcified in decalcifying solution (Sigma‐Aldrich) at room temperature for 7 days. The samples were paraffin‐embedded and sectioned into 5 μm thick sections. Deparaffinized sections were stained with Hematoxylin and Eosin (H&E) staining.
For immunohistochemistry, deparaffinized sections were rehydrated and subjected to antigen retrieval at 37°C for 30 min. The samples were protein blocked with a serum‐free blocking solution at room temperature for 1 h, then, incubated with anti‐OPN (1:100 dilution, Abcam, MA, USA), anti‐OCN (1:50 dilution, Santa Cruz, CA, USA), anti‐DSPP (1:50 dilution, Santa Cruz, CA, USA), and ani‐DMP‐1 (1:500 dilution, Santa Cruz, CA, USA) primary antibodies at 4°C overnight. The samples were incubated with the secondary biotinylated anti‐goat antibody (BA‐5000, Vector Laboratories) for 30 min. Streptavidin‐conjugated horseradish peroxidase (SA‐5704, Vector Laboratories) was added for 30 min, and the samples were stained with 3,3′‐Diaminobenzidine (DAB). Then, Gill's hematoxylin was used to counterstain the cell nuclei. The stained sections were visualized under the light microscope and the positive areas of OPN, OCN, DSPP, and DMP‐1 were measured using Image J software.