Eclipse e1000 light microscope

Histology and immunohistochemistry (IHC) were carried out on paraformaldehyde-fixed histological IP and NU tissue sections (n = 8 and 9 respectively). Haematoxylin and eosin staining was performed according to standard protocols. IHC staining was carried out using EnVision peroxidase/DAB+ detection system (Dako, Stockport, UK) and specific anti-human antibodies (Supplementary Table S1). In cases where antigen retrieval was required, this was performed by heating sections in boiling 10 nM citrate buffer (pH 6.0) in a 900 W microwave oven for 10 min. All images were captured using an Eclipse E1000 light microscope (Nikon). For measurement of average blood vessel size, up to six photomicrographs (dependent on tissue size) were taken at 200× magnification following CD31 labelling. Measurement of blood vessel areas was then performed blinded using ImageJ free access image processing and analysis software available from http://rsb.info.nih.gov/ij/. The number of vessels was counted in at least 20 different random fields of each experimental group, as previously described [48 (link)]. Analysis of CD45, stromal-derived factor 1 (SDF1), vascular endothelial growth factor 1 (VEGF), and bone morphogenetic protein 2 (BMP-2) expression was performed using NIS elements BR (Nikon Instruments Inc., Melville, USA ) image analysis software.

Donor-matched IC-BM and VB-BM MSCs (expanded for three passages) were seeded at 2.5x10⁵ per conical tube for the chondrogenic assays. Triplicates were used for quantitative measurements of glycosaminoglycan (GAG) levels and duplicates for the GAG staining. As previously described [32 (link)], cells were cultured in chondrogenic media, consisting of high glucose DMEM (Thermo Fisher Scientific) supplemented with; l-ascorbic acid-2-phosphate, sodium pyruvate, proline, Bovine serum albumin, penicillin/streptomycin, dexamethasone, insulin-transferrin-selenium (all from Sigma-Aldrich) and TGFβ3 (R&D Systems, Abingdon, UK). Following 21 days of culture, cell pellets were digested in papain solution (100mM Sodium Phosphate Buffer supplemented with 5mM Na₂EDTA, 10mM l-cysteine and papain, all from Sigma) and the levels of GAG were measured using a Blyscan™ kit (Biocolor Life Sciences, Co Antrim, Ireland) as per manufacturer instructions. For the GAG staining, the cells were treated with 1% toluidine blue (Sigma-Aldrich) then the images for GAG-stained cells were captured using an Eclipse E1000 light microscope (Nikon, Surrey, UK).