Proteinase k

Chondrocyte cell death was evaluated using an in situ cell death detection kit (KeyGEN Biotech, Nanjing, China) based upon the presence of DNA fragmentation. Briefly, sagittal cartilaginous tissue sections from knee joints were treated with 20 μg/ml proteinase K (Dako, Glostrup, Denmark) for 15 min, after which apoptotic chondrocytes in the articular cartilage were labeled. Numbers of apoptotic cells were then quantified in five randomly selected high-power fields (×100) of view per sample in tissues from each group.

Native and decellularised zinc fixed and paraffin embedded tissue sections (from n = 6 human aortas) were stained with monoclonal antibodies against collagen I (Millipore; IgG isotype, polyclonal, 1:500 dilution), collagen III (Millipore; IgG1 isotype, clone IE7-D7, 1:100 dilution), collagen IV (Dako; IgG1 isotype, clone PHM-12, 1:200 dilution), von Willebrand factor (Dako; IgG isotype, polyclonal, 1:400 dilution), fibronectin (Vector; IgG1 isotype, clone 568, 1:150 dilution) and laminin (Sigma; IgG1 isotype, clone LAM-89, 1:1000 dilution). Immunolabeling was carried out using an Ultravision detection system (Thermo Scientific). Antigen retrieval was employed using proteinase K (Dako; room temperature for 20 min—collagen III, von Willebrand factor, fibronectin and laminin), proteinase K followed by trypsin digestion (0.05% w/v trypsin 0.52 mM EDTA.4Na in HBSS with phenol red (Sigma) for 30 s) (collagen IV) and Vector antigen unmasking solution citric based (collagen I). Hydrogen peroxide (Sigma) was used to block endogenous peroxidase activity. Isotype control antibodies (IgG and IgG1; Dako; used at the same concentrations as the test antibody) and omission of the primary antibody served as negative controls. Sections were viewed using Kohler and all images were captured digitally using Zen Blue Pro (Zeiss).

Formalin-fixed synovial tissues from three patients (one male of 60-year-old, two females 54- and 59-year old) undergoing revision of total hip arthroplasty were embedded in paraffin and 3 μm sections were used for immunohistochemistry staining (IHC). All samples didn’t show any clinical signs of local infection with the level of serum CRP below 0.07 mg/dl. Control tissue was collected from patient of hip osteoarthritis (female 64-year-old) undergoing primary hip arthroplasty. Sections were deparaffinized, treated for 5 min with proteinase K (Dako, CA, USA) for antigen retrieval, and then blocked with horse serum for 1 h. Sections were incubated with primary antibodies to myeloperoxidase (MPO: JM10–58, 1:200 dilution, Novus Biologicals, USA), neutrophil Elastase (ab68672, 1:200 dilution, Abcam, Cambridge, UK), CD68 (KP1, clone PG-M1, M0876: 1:200 dilution, Dako Agilent, USA), and AnxA1 (GTX113329; 1:200 dilution, GeneTex, CA, USA) overnight and then washed three times with tris-buffered saline buffer (TBS). The signal was amplified with Vectastain Elite ABC kit for detection of horseradish peroxidase (HRP) (Vector Laboratories, Burlingame, USA) followed by counterstaining with hematoxylin for detecting cellular nuclei.

Skin samples were paraffin-embedded, and sections were dewaxed in xylene and rehydrated in graded alcohols. Haematoxylin and eosin (HE) staining or Masson's trichrome staining was performed as described previously [15] (link).
For immunostaining, antigens were retrieved by incubation with proteinase K (DAKO) for 6 minutes. Endogenous peroxidase activity was inhibited, after which sections were blocked with 3% bovine serum albumin (BSA, Sigma) for 20 minutes and then reacted with the primary antibodies for ET-1 (1∶250, Peninsula Laboratories), myeloperoxidase (1∶100, Thermo), F4/80 (1∶100, Abcam), or CD3 (1∶100, Serotec) overnight at 4°C. After excess antibody was washed out with PBS, sections were incubated with appropriate HRP-labeled secondary antibody (Nichirei) for 60 minutes at 20°C. The reaction was visualized by diaminobenzidine substrate system (Dojin). Slides were counterstained with Mayer's haematoxylin, and examined under a light microscope (OLYMPUS).

For LSAB (Labeled (Streptavidin-Biotin) staining, paraffin-embedded, formalin-fixed tissue sections were deparaffinized and re-hydrated in xylene, ethanol and TBS. Endogenous peroxidases (15 min incubation in 3% H₂O₂) and endogenous biotin (Avidin/Biotin Blocking Kit, Vector Laboratories, Burlingame, CA) were blocked. For antigen retrieval, sections were incubated in Proteinase K (DAKO, Carpinteria, CA) for 7 min, blocked with 1% BSA in PBS and incubated with the primary rabbit anti-Nod1 (1:200, Imgenex IMG 5739), anti-Nod2 (1:600, Santa Cruz, sc 56168), anti-RIPK2 (1:200, Abcam, ab 75257), anti-caspase3 antibody (1:100, cell signaling) and anti-cleaved (c) -Caspase3 antibody (1:100, Cell Signaling) in PBS overnight. Washed in 0.1% BSA, washed in PBS and treated with biotin sheep anti-rabbit secondary antibodies (DAKO) and the AEC peroxidase substrate kit (Vector Laboratories) according to the manufacturer’s instructions. Positive controls were performed according to the protocol. Negative controls without antibody were used for every experimental epitope target.