Recombinant Anti-SOX9 antibody [EPR14335] (ab185230)

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The articular cartilage and subchondral bone specimens were analyzed with immunohistochemical analysis by anti-human specific nuclei antigen antibody, anti-rat TGF-β1 (transforming growth factor β1), anti-rat IGF-1 (Insulin-like growth factor 1), anti-rat type II collagen, TUNEL activity, MMP-13, RUNX-2, SOX-9 and Collagen Xα1. The harvested specimens were fixed in 4% PBS-buffered formaldehyde for 48 hours and decalcified in PBS-buffered 10% EDTA solution. Decalcified tissues were embedded in paraffin wax. The specimens were cut longitudinally into 5 μm thick sections and transferred to poly-lysine-coated slides. Sections of the specimens were immunostained with specific reagents for anti-human specific nuclei antigen antibody, anti-rat TGF-β1, IGF-1 and type II collagen (Santa Cruz Biotechnology Inc, CA, USA), MMP-13, RUNX-2, SOX-9 (Abcam, Cambridge, UK), and Collagen Xα1 (Gene Tex, Inc., USA) to identify the location of WJMSCs, cartilage, and osteogenesis markers in bone remodeling and cartilage regeneration of rats. The immuno-reactivity in specimens were demonstrated using a horseradish peroxidase (HRP)-3’-, 3’-diaminobenzidine (DAB) cell and tissue staining kit (R & D Systems, Inc. Minneapolis, MN, USA). In Situ Cell Death Detection Kits (Roche Diagnostic, Mannheim, Germany) accomplished the TUNEL analysis following a manufacture instruction. TUNEL color stains were performed by using NBC/BCIP substrate (Sigma-Aldrich, St. Louis, MO, USA). The immuno-activities were quantified from five areas in three sections of the same specimen using a Zeiss Axioskop 2 plus microscope (Carl Zeiss, Gottingen, Germany). All the images of each specimen were captured using a Cool CCD camera (SNAP-Pro c.f. Digital kit; Media Cybernetics, Silver Spring, MD, USA). Images were analyzed using an Image-Pro® Plus image-analysis software (Media Cybernetics, Sliver Spring, MD, USA). The percentage of each area was counted by immuno-labeled positive cells over the total cells as the results.

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