Anti runx2 antibody

Proteins were extracted from the harvested hind limbs of 3-week-old rats. Isolated femurs and tibias were snap-frozen by adding liquid nitrogen on a mortar and homogenized using a pestle. Pulverized samples were incubated in the red blood cell lysis buffer (155 mM NH₄Cl, 140 mM NaHCO₃, and 0.1 mM EDTA, pH 7.3) for 10 min, and supernatants were removed by centrifuge. Pellets were lysed in ice-cold RIPA buffer containing protease inhibitor cocktails (Roche Applied Science). A controlled amount of proteins was separated using SDS-PAGE and transferred onto a PVDF membrane using an electroblot. After blocking with 5% milk TBS-T, the blots were probed using primary antibodies (anti-ZIP8 antibody, Pierce Biotechnology, Inc.; anti-Runx2 antibody, Sigma-Aldrich Co.; anti-β-actin antibody, Cell Signaling Technology, Inc.; and anti-Collagen-1 antibody, Santa Cruz Biotech.) and followed by a horseradish peroxidase-conjugated secondary antibody. Chemiluminescent reagents were used to detect immunoreactive proteins, and the membrane was exposed to X-ray films.

Additional 3-day-old male pups exposed to IH (n = 2) and room air (n = 2) conditions were euthanized using CO₂ and fixed in 10% neutral buffered formalin (Sigma-Aldrich Co.) for 7 days. Hind limbs were isolated and outsourced to the Pathology and Laboratory Medicine Services at UCLA for immunohistochemistry staining. The specimens were decalcified for paraffin embedding. Comparable sections of paraffin embedded specimens were processed with anti-Runx2 antibody (Sigma-Aldrich Co.) on the slides in accordance with the manufacturer’s guidelines. All sections were visualized using diaminobenzidine reaction and counterstained with hematoxylin. The corresponding sections were stained with Masson Trichrome (Sigma-Aldrich Co.), in accordance with the manufacturer’s protocol. Histological evaluations were performed with Aperio ImageScope (Leica Microsystems, Inc.) on digital images.