Bright ot5000 cryostat

1 × 10⁶ cells were cultured as pellet culture in StemPro Osteocyte/Chondrocyte basal medium (Life Technologies) supplemented with StemPro Chondrogenesis supplement (Life Technologies) in a humidified incubator at 37°C and 5% CO₂. Fresh medium was provided every 2–3 days. After 21 days, medium was aspirated and cell pellets were washed with PBS before fixation in 4% PFA for 60 min. After 3 washings steps with ddH₂O, Alcian Blue staining solution was added [0.1 mg/ml Alcian Blue dissolved in 6 parts EtOH and 4 parts of acetic acid (Sigma-Aldrich)] and incubated over night at room temperature under exclusion of light. Cell pellets were embedded in O.C.T compound (TAAB Laboratory Equipment Ltd., Reading, UK) and frozen in super-cooled ethanol at −80°C. Cryostat sections (10 μm) were prepared (Bright OT5000 cryostat), mounted on poly-lysine-coated glass slides (VWR) and visualized using a Nikon TiE microscope (Nikon UK Ltd., Kingston upon Thames, UK).

The mucoperiosteum was embedded in O.C.T compound (TAAB Laboratory Equipment Ltd., Reading, UK) and quick-frozen in super-cooled ethanol (−80°C). Cryostat sections (10 μm, Bright OT5000 Cryostat) were cut and mounted on poly-lysine-coated glass slides (VWR International Ltd., Lutterworth, UK) and processed for immunocytochemistry as stated below.