Alexa fluor 488 conjugated goat polyclonal anti mouse immunoglobulin g igg

Immunostaining for ECM deposition in cryosectioned samples was performed using primary monoclonal antibodies for type I, II, and X collagen, aggrecan, and α-smooth muscle actin (α-SMA). Antigen retrieval was performed for all sections by incubating in 20 µg/ml proteinase K (Sigma-Aldrich) for 10 minutes at 37°C immediately prior to staining. Samples for aggrecan and collagen X immunostaining were deglycosylated with 0.75U/mL chondroitinase ABC (Sigma-Aldrich) for 1.5 hours at 37°C. Samples were blocked with Image-iT FX Signal Enhancer (Life Technologies, Carlsbad, CA) and incubated with the primary antibodies (for dilutions & vendor information, see Supplementary Table 2) overnight at 4°C. Secondary antibody binding with Alexa Fluor 488-conjugated goat polyclonal anti-mouse immunoglobulin G (IgG, Molecular Probes, Carlsbad, CA) or IgM (Molecular Probes) was performed at room temperature for 1 hour. The samples were stained with Hoechst (Sigma-Aldrich) to visualize the nuclei. Isotype controls were similarly stained using a monoclonal mouse IgG1(Abcam) or IgM (Abcam) isotype antibody (minimal signal was observed with isotype controls; data not shown).

For histological analysis of ATDC5 spheroids, spheroids were imbedded in Histogel, sectioned at 10 µm sections and stained with Safranin-O according to standard protocols. Immunostaining for ECM deposition was performed using primary antibodies for collagen type II (Abcam). Antigen retrieval was performed by incubating in 20 µg mL⁻¹ proteinase K (Sigma-Aldrich) for 10 minutes at 37°C, blocked with 1.5% goat serum (Fisher Scientific), and incubated with the primary antibodies at a 1:20 dilution overnight at 4°C. Secondary antibody binding with Alexa Fluor 488-conjugated goat polyclonal anti-mouse immunoglobulin G (IgG, Molecular Probes) was performed at room temperature for 1 hour. Samples were counterstained with Hoechst (Sigma-Aldrich) to visualize the nuclei.
For alcian blue and alizarin red staining in transwell culture, cell monolayers were rinsed in ddH₂O and fixed in 95% ice cold methanol for 30 minutes. Cells were stained with either 1% alcian blue 8GX (Sigma-Aldrich) or 2% alizarin red (Sigma-Aldrich) pH 4.2 in 0.1 M HCl for 1 hour. Cells were then rinsed with ddH₂O and imaged. Stain was extracted by incubating stained cells with solutions of 6 M guanidine-HCl (Sigma-Aldrich, alcian blue) or 5% SDS in 0.5M HCl (alizarin red) for 6 hours. Absorbance was measured at 630 and 405 nm for alcian blue and alizarin red extractions, respectively.