Igg1 control antibody

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).

Synovial biopsies were fixed in 10% neutral-buffered formalin solution followed by paraffin embedding. Synovial tissue sections, 3 μm thick, were heated for 30 minutes at 60°C, deparaffinized in xylene, and rehydrated in alcohol and deionized 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 deionized water, pH 6.0) in a pressure cooker. Slides were washed in PBS for 5 minutes. Nonspecific binding was blocked using 10% casein in PBS for 30 minutes. Primary antibodies aPD-1 (Abcam, clone: NAT105) and aCD19 (Thermo Fisher Scientific, clone: JF100-06) were incubated on sections for 2 hours at room temperature. An IgG1 control antibody (Dako) was used as a negative control. Slides were washed in PBS/Tween followed by 1-hour incubation at room temperature with secondary Cy2 (catalog 115-225-146) and Cy3 (catalog 111-165-144) AffiniPure secondary antibodies (Jackson ImmunoResearch). Slides were washed with PBS/Tween and PBS, before counterstaining of nuclei with DAPI (MilliporeSigma) and cover slide mounting with ProLong Gold Antifade (Thermo Fisher Scientific). Stained cells were visualized with a Leitz DM40 microscope (Leica Microsystems), and images were captured using the AxioCam system and AxioVision 3.0.6 software (Carl Zeiss Inc).