Sections (5 μm each) were cut and mounted on StarFrost adhesive glass slides (Knittelgläser, Braunschweig, Germany). Sealed slides were stored at −80°C until further use. LN tissue sections were stained using mouse monoclonal antibodies against T cells (anti-CD3, clone SK7; Becton Dickinson, Breda, the Netherlands), B cells (anti-CD22, clone RFB4; Millipore, Amsterdam, the Netherlands) and plasma cells (anti-CD138, clone MI15; Dako, Heverlee, Belgium).
Staining was performed using a three-step immunoperoxidase method to detect bound anti-CD138 antibodies, as described previously [17 (link)]. For anti-CD3 and anti-CD22, we used a two-step immunoperoxidase method with a secondary polymer—horseradish peroxidase—conjugated anti-mouse antibody (EnVision+ System; Dako). As a negative control, irrelevant isotype-matched immunoglobulins, instead of the primary antibody, were applied to the sections. Staining was analysed by digital image analysis in a blinded fashion using a Syndia algorithm on a Qwin-based analysis system (Leica, Cambridge, UK) as described previously [18 (link)]. The number of positive cells was calculated for each section as the number of positive cells per square millimetre of tissue.
Staining was performed using a three-step immunoperoxidase method to detect bound anti-CD138 antibodies, as described previously [17 (link)]. For anti-CD3 and anti-CD22, we used a two-step immunoperoxidase method with a secondary polymer—horseradish peroxidase—conjugated anti-mouse antibody (EnVision+ System; Dako). As a negative control, irrelevant isotype-matched immunoglobulins, instead of the primary antibody, were applied to the sections. Staining was analysed by digital image analysis in a blinded fashion using a Syndia algorithm on a Qwin-based analysis system (Leica, Cambridge, UK) as described previously [18 (link)]. The number of positive cells was calculated for each section as the number of positive cells per square millimetre of tissue.
