Alexa second antibody

The dissected samples were fixed in 4% PFA overnight and decalcified by 10% EDTA (pH 7.4) at 4℃, followed by 30% sucrose dehydration and cryosection with optimum cutting temperature compound (O.C.T. compound, Tissue-Tek) at the thickness of 10µm. Sections were stained by: mouse anti-nestin monoclone antibody (1:100, EMD Millipore, CA, USA), and mouse anti-biglycan monoclone antibody LF-159 (1:1000, Kerafast, MA, USA). The immunofluorescent signals were detected with corresponding Alexa second antibody (Thermofisher, 1:200) at room temperature for 2 hours.

Mandibular condyles were fixed in 4% paraformaldehyde and decalcified at 4 °C. Samples for cell lineage tracing were dehydrated with sucrose and embedded in OCT followed by CryoJane frozen sections as previously described 30 (link). Samples for histological staining were embedded in paraffin, sectioned, and stained with safranin O (proteoglycans) or toluidine blue stain 31 (link). Immunostaining were proceeded as previously described 32 with the following antibodies: rabbit anti-aggrecan antibody (Abcam; 1:400), anti-collagen II mouse monoclonal antibody (Santa Cruz Biotechnology; 1:50), rabbit anti-collagen X antibody (Abcam; 1:400), rabbit anti-DMP1 antibody (provided by Dr. Chunlin Qin at Texas A&M University, 1:400), rabbit anti-Runx2 antibody (Abcam; 1:200), goat anti-sclerostin antibody (R&D; 1:400), rabbit anti-collagen I antibody (Abcam; 1:100), anti-osteopontin mouse monoclonal antibody (Santa Cruz Biotechnology; 1:100), or anti-β-catenin mouse monoclonal antibody (DSHB; 1:100). The immunohistochemistry experiments were detected with a 3, 3-diaminobenzidine kit (Vector Laboratories, Burlingame, CA). The immunofluorescent signals were detected with corresponding Alexa second antibody (Thermofisher, 1:200) at room temperature for 2 hours.