CXCL13 expression in situ by CD68⁺ macrophages in lymph nodes from CLL patients. Paraffin-embedded sections of lymph nodes from CLL patients were incubation with anti-CD68 and anti-CXCL13 mAbs. For simultaneous detection of CD68 and CXCL13, a biotin-free system was used that is based on peroxidase with DAB as chromogen (brown) for CD68 detection and alkaline phosphatase with permanent red chromogenic substrate for detection of CXCL13. Images were visualized using a Zeiss Axiovert 135 microscope, C-Apochromat objectives, an AxioCam ICc3 camera, and Axioplan 2 imaging software (all from Carl Zeiss, Jena, Germany) and processed using Canvas 9 software (ACD Systems, Victoria, BC). (A) A lymph node section at low magnification (200×) that depicts CD68⁺ cells scattered throughout the displayed section that coexpress CXCL13. (B,C) Higher magnification that depicts coexpression of CD68 (brown, perinuclear) and CXCL13 (red) at 630×.