Sc 20095

A semiquantitative analysis of differentiation was performed on cell-seeded hydrogels via Western blotting as described previously using the following antibodies that were diluted in 3% BSA in TBST buffer: aggrecan (1 : 1000, sc-16492, Santa Cruz Biotechnology, USA), Col II (1 : 5000, ab34712, Abcam, USA), and Sox-9 (1 : 1000, sc-20095, Santa Cruz Biotechnology, USA) antibodies and goat anti-mouse horseradish peroxidase-conjugated secondary antibody (1 : 2000, CW0102, Cwbiotech). And the protein level was quantified and normalized to GAPDH bands by densitometry in Quantity One software (version 4.6.2, Bio-Rad, n = 3).

Immunohistochemistry on paraffin sections was performed as previously described [9 (link)]. Briefly, skin samples were fixed in 4% buffered paraformaldehyde and embedded in paraffin. For immunohistochemistry, antigen retrieval was performed in citrate buffer (pH 6.0) for 10 minutes at 110°C in HistoPro (Rapid Microwave Histoprocessor, Milestone, USA). The following primary antibodies were used: anti-Sox10 (goat, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Sox10 (mouse, 1:200, R&D), anti-Sox9 (rabbit, 1:100, sc-20095, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Sox9 (rabbit, 1:100, ab36748, Abcam), anti-Sox9 (M00006662, Abnova), anti-Sox9 (AB5535, Millipore), anti-Sox9 (GTX 109661, GenTex), anti-MITF (mouse, clone 6D3, 1:500) was a kind gift from Heinz Arnheiter (NIH, USA). Images were captured with a Leica DMI 6000B Microscope and using LAS AF (Leica Application Suite Advanced Fluorescence) software. For whole mount X-Gal staining, skin samples were fixed with 4% buffered paraformaldehyde, washed with PBS and subjected to X-Gal staining solution overnight at 37°C. After several washing steps, tissue was paraffin embedded and sectioned. 5 μm thick sections were further counterstained with eosin solution and mounted.