Gill s haematoxylin no 3

Both femoral condyles were fixed (4% paraformaldehyde), decalcified (20% EDTA, pH 7.4), and embedded in paraffin. Proteoglycan staining by means of safranin-O (SafO) allowed for histopathological assessment. In short, dewaxed and rehydrated sections were stained with SafO (Fisher Scientific, Schwerte, Deutschland) and Fast Green (Sigma-Aldrich, Taufkirchen, Germany), followed by a final staining of the cell nuclei by Gill’s haematoxylin No. 3 (Sigma-Aldrich) and documentation with an Axioskop 2 mot plus (Zeiss, Oberkochen, Germany). The corresponding scoring criteria and their respective specifications, which were principally derived from Laverty et al. [11 (link)], are depicted in the supplementary material S2. The scoring was performed independently by two observers (interclass correlation: 0.81).

For immunohistochemistry (IHC), 3.5 μm-thick paraffin-embedded sections of the pellets from the chondrogenic redifferentiation were dewaxed, rehydrated and pre-digested for 30 min at 37°C for antigen retrieval with pepsin (1 mg/ml in 0.5 M acetic acid) (porcine gastric mucosa) (Sigma Aldrich), in case of collagen type II staining. Sections were treated with 3% hydrogen peroxide before starting the staining with the Dako labeled streptavidin-biotin (LSAB) 2 system-horseradish peroxidase (HRP) kit (Dako) and antibodies against collagen type II (AF5710; Acris, Hiddenhausen, Germany, and MA5-13026; Invitrogen). For histological staining of proteoglycans, Safranin O (Fisher Scientific) was used. In all samples, a final staining of cell nuclei by Gill’s haematoxylin No. 3 (Sigma-Aldrich) was performed.