Poly l lysine coated microscope slides

Dulbecco's modified Eagle medium (DMEM Cat. No 41966) and non-essential amino acids (NEAA) were from Life Technologies (Bleiswijk, the Netherlands). Foetal Bovine Serum (FBS, batch F7524-500ML / lot BCBV7611) was from Sigma Aldrich / Merck. Antigen retrieval citrate buffer, RPMI-1640 medium, poly-L-lysine coated microscope slides and SnakeSkin Dialysis tubing were from Thermo Fisher Scientific (Breda, the Netherlands). Disposable biopsy punches were from Amstel Medical (Amstelveen, the Netherlands) Trypsin-EDTA (0.25 %) and penicillin/ streptomycin (P/S) were from Lonza (Breda, the www.ecmjournal.org Netherlands). Human bone marrow (healthy male subject, 24 years old) was from Lonza (Walkersville, MD, USA). The human buffy coat was from Sanquin (Nijmegen, the Netherlands). Lymphoprep™ was from Axis-Shield (Oslo, Norway). MACS ® Pan Monocyte Isolation Kit was from Miltenyi Biotec (Leiden, the Netherlands). Recombinant human basic fibroblast growth factor (bFGF), macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL) were from PeproTech (Rocky Hill, NJ, USA). Bombyx mori L. Silkworm cocoons were from Tajima Shoji Co., LTD. (Yokohama, Japan). Thin bleach was from the local grocery store. All other substances were of analytical or pharmaceutical grade and obtained from Sigma Aldrich / Merck (Zwijndrecht, the Netherlands).

Constructs were fixed in 10 % neutral buffered formalin (24 h at 4 °C), dehydrated in graded ethanol solutions and embedded in paraffin, bisected through the centre, cut into 6 µm thick sections and mounted on poly-L-lysine coated microscope slides (Thermo Fisher Scientific, Breda, the Netherlands).
Sections were stained with alizarin red to identify mineralisation, picrosirius red to identify collagen, alcian blue to identify glycosaminoglycans (GAGs) and H&E for cell location. Figures show representative images of all the samples assessed.