Gill s hematoxylin no 3

For IHC, paraffin-embedded sections (3.5 μm) were dewaxed and rehydrated. Antigen retrieval was performed by incubation in sodium citrate buffer pH 6.0 at 65 °C overnight. After cooling, unspecific antigens were blocked with the DAKO blocking reagent (Dako, Glostrup, Denmark), followed by overnight incubation with the 1:250 diluted rabbit monoclonal antibody [EPR9514] against human p-MLKL (phospho S358; abcam, Cambridge, UK), human cleaved caspase 8 (NB100-56116; NOVUS Biologicals, Centennial, CO, USA) or RIPK3 (GTX107574; GeneTex, Irvine, CA, USA) at 4 °C. Sections were treated with 3% hydrogen peroxide before starting the staining with the Dako LSAB2 System-HRP kit (Dako, Glostrup, Denmark). In all samples a final staining of cell nuclei by Gill’s hematoxylin No 3 (Sigma-Aldrich) was performed. In case of cleaved CASP8 and p-MLKL, the percentage of positive cells was quantified by manual counting of the stained sections.

To evaluate the content of PG within the cartilage tissue, appropriate histological analysis was performed exemplarily (n = 1) 14 days after trauma. In short, explants were fixed (4% paraformaldehyde) and embedded in paraffin. Dewaxed and rehydrated sections (3.5 µm) were stained with SafO (Thermo Fisher Scientific, Schwerte, Germany) and Fast Green (Sigma-Aldrich), followed by a final staining of the cell nuclei by Gill’s hematoxylin No. 3 (Sigma-Aldrich) and documentation with an Axioskop 2 mot plus (Zeiss, Oberkochen, Germany).