Anti aggrecan antibody

All materials and chemicals utilised in the experiments were of laboratory grade. Advanced DMEM/F12 and other materials for cell culture were acquired from Thermo Fisher Scientific (Waltham, MA, USA). Heat inactivated foetal bovine serum was purchased from Gibco (by Thermo Fisher Scientific, Waltham, MA, USA). Streptomycin, penicillin, L-glutamine, phosphate-buffered saline (PBS), bovine serum albumin (BSA), Tween 20 and trypsin-EDTA were bought from Sigma-Aldrich (Merck KgaA, Darmstadt, Germany). Anti-Aggrecan antibody, Anti-Collagen 1 antibody, Anti-Collagen 2 antibody, Rabbit Anti-Mouse IgG H&L secondary antibody Alexa Fluor 488, Goat anti-rabbit IgG (H+L) secondary antibody Alexa Fluor 594, CytoPainter Phalloidin-iFluor 555 Reagent and Fluoroshield Mounting Medium with DAPI were from AbCam (Cambridge, UK) and Fixation solution (5×) from Millipore (Merck, Millipore, Darmstadt, Germany). All other substances were acquired from standard commercial suppliers.

For immunofluorescence staining, ADSCs were seeded on glass slides (Ø 13 mm) at a previously mentioned ratio. After 14 days of culturing, the differentiation medium was removed and cells were fixed in 4% PFA (Serva, Germany) for 10 min. After fixation, cells were washed with 1× PBS three times. Then for permeabilization, 0.1% Triton X-100 (Serva, Germany) was added and kept for 10 min and then cells were washed with 1× PBS three times. To reduce non-specific binding, cells were blocked with 1% bovine serum albumin (BSA)/PBS. To detect expressed protein of interest for specific lineage commitment, cells were incubated at overnight at 4 °C with anti-aggrecan antibody (Abcam, USA Cat No: Ab36861) at a 1:100 dilution in 0.1% BSA/PBS, anti-PPAR gamma antibody (Abcam, USA Cat No: Ab36861) at a 1:200 dilution in 0.1% BSA/PBS and anti-osteopontin antibody (Abcam, USA Cat No: Ab8448) at a 1:200 dilution in 0.1% BSA/PBS. Then to detect primary antibodies goat anti-rabbit IgG H&L Alexa Fluor 488 (Abcam, USA, Cat No: Ab150077) was used as a secondary antibody at a ratio 1:200 in 0.1% BSA/PBS. Then cover glass were mounted with Fluoromount W (Serva, Germany). Then cells were imaged under up right fluorescence light microscope Olympus BX43 (Olympus, UK).