Dako chem mate envision kit

Paraffin-embedded tissue microarray was deparaffinized in xylene and ethanol (100%, 90%, and 75%, respectively) and rehydrated. The microarray was boiled in 5,000 mL EDTA (pH = 8.0) for 5 min at high pressure, and endogenous peroxidase was quenched using 0.5% hydrogen peroxide. After washing three times with PBS, the microarray was blocked with 7% normal goat serum and incubated with anti-DPYSL2 antibody (1:100) overnight at 4°C. The results were visualized using a Dako Chem-Mate EnVision kit (K500711; Dako, Agilent Technologies, Santa Clara, CA, United States) and were assessed by two independent pathologists in a blinded manner. The intensity of the staining was scored as previously described (Zou et al., 2019 (link)).

Immunohistochemistry analysis was performed using 3 μm paraffin synovial tissue sections and the Dako ChemMate Envision Kit (Dako, UK) in RA (n = 25) and OA (n = 12). Sections were baked for 30 mins at 90 °C, deparaffinised in xylene and rehydrated in alcohol and deionised water. Antigen retrieval was performed by heating sections in antigen retrieval solution (15 ml of 1 M sodium citrate and 15 ml of 1 M citric acid in deionised water, pH 6.0) in a pressure cooker. Slides were washed in PBS for 5 mins. Non-specific binding was blocked using 10% casein in PBS for 20 mins. GAPDH (Trevigen, Gaithersburg, MD), PKM2 (Abgent, CA) and GLUT-1 (Abcam, UK) (Santa Cruz Biotechnology, CA) primary antibodies were incubated on sections for 2 hrs at room temperature. An IgG1 control antibody (Dako) was used as a negative control. Slides were incubated for 1hr with horseradish peroxidase–conjugated secondary antibody (Dako). Colour was developed in diaminobenzidine solution (1:50; Dako) and counterstained with hematoxylin. Slides were mounted in Pertex media and analysed using a well-established semi-quantitative scoring method (0–4) and scored separately for perivascular/vascular, lining layer and sub-lining layer regions74 (link).

Four-micrometer-thick sections from paraffin-embedded blocks were cut onto precoated slides, followed by deparaffinization, rehydration, and antigen retrieval as previously described [14 (link), 15 (link)]. Endogenous peroxidase was blocked per the manufacturer's protocol (Dako, Carpinteria, CA). The slides were incubated with an anti-CSF-1 monoclonal antibody (MABF191, Merck Millipore) at a 1 : 200 dilution at 4°C for 1 h. Primary antibodies were detected using the Dako ChemMate EnVision Kit (K5001, Dako, Carpinteria, CA). Finally, the slides were counterstained with hematoxylin and investigated by light microscopy.