Aminoethylcarbazole color reaction

The synovial biopsies were embedded in paraffin, sectioned, and subjected to antigen retrieval by cooking when required. The slides were subsequently stained with an automated immunostainer (TechMate 500 Plus; Dako, Cambridge, UK) using the following monoclonal antibodies: anti-CD3 (clone PS1; Novocastra, Newcastle, UK), anti-CD20 (clone L26; Dako), anti-CD68 (clone KP-1; Dako), and anti-CD138 (clone B-B4; Santa Cruz Biotechnology, Inc., San Diego, CA, USA. As a negative control, the primary antibodies were substituted by isotype- and concentration-matched control antibodies. The primary antibodies were subsequently detected by an avidin-biotin-peroxidase-based method (Envision System; Dako) and an aminoethylcarbazole color reaction (Sigma-Aldrich, St. Louis, MO, USA) as described previously in detail (9 (link)). Finally, the slides were counterstained with hematoxylin. The stained slides were scored by digital image analysis by an independent observer (RC) who was blinded to diagnosis and clinical data. Each stained slide in its entirety was scored by dividing it in different regions. Within each region, the number of stained cells per area as well as the percentage of stained cells were measured in at least 20 high-power fields using the AnalySIS^® Imaging processing program (Olympus^®) as described previously in detail (9 (link)).

ST were routinely fixed and embedded in paraffin. Deparaffinized sections were cooked to perform antigen retrieval when required. Slides were subsequently stained with an automated immunostainer (TechMate 500 Plus; Dako, Cambridge, UK) using the following monoclonal antibodies: anti-CD3 (clone PS1; Novocastra, Newcastle, UK) for T-lymphocytes, anti-CD20 (clone L26; Dako) for B-lymphocytes, anti-CD68 (clone KP-1; Dako) for CD68 + macrophages, anti-CD117 (rabbit anti-human polyclonal antibody; Dako) for mast cells, anti-Hsp47 monoclonal antibodies (IgG2b M16.10A1 clone; Assay Designs) for synovial fibroblasts, anti-CD31 (clone JC70A; Dako) for endothelial cells, and anti-basic fibroblast growth factor (bFGF) (polyclonal SC-79, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-CXCL12 (clone K15C [22 (link)]) for angiogenic markers, which were significantly increased in serum from these patients in our previous study [20 (link)]. As a negative control, the primary antibodies were substituted by isotype-matched and concentration-matched control antibodies. The primary antibodies were subsequently detected by an avidin-biotin-peroxidase-based method (Envision System; Dako) and an aminoethylcarbazole color reaction (Sigma-Aldrich, St. Louis, MO, USA) as previously described [23 (link)]. Finally, the slides were counterstained with hematoxylin.