H&E staining and IHC were performed on formalin-fixed and paraffin-embedded LNs, which included nine enlarged and five non-enlarged LNs. All LNs were dissected transversely in the middle of the longitudinal axis and serially sectioned. The primary antibodies used were anti-CD20 (Cell Signaling Technology, 48750T, 1: 100) for identifying B cells, anti-CD68 (Cell Signaling Technology, 76437T) for identifying macrophages, anti-CD3 (Servicebio, GB11014) for identifying T cells, and anti-Ki67(Cell Signaling Technology, 9449T) for identifying proliferating cells (18 (link)). Diluents without primary antibodies were used as negative controls. Staining was visualized using the Dako REAL™ EnVision™ Detection System followed by counterstaining with hematoxylin. Images were captured using a digital camera under a light microscope (VS120; Olympus). The number of germinal centers (GCs) observed was counted in one HE-stained section. The proportions of CD68- and Ki67-positive cells in the LNs were calculated as positive cells versus total cells in at least five randomly selected areas under ×1000 magnification of microscopic fields.
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