CCL21 mRNA is not detected in HEVs of normal human lymphoid tissues but is expressed in lymphatic endothelium and perivascular cells. In situ hybridization for CCL21 mRNA (red) and subsequent immunofluorescence staining for peripheral lymph node addressin (PNAd) with the monoclonal MECA-79 antibody (green) in normal mouse and human lymphoid tissues. Insets in left column display negative sense controls. Left and middle columns are light microscopic exposures in which PNAd expression is not visualized. Right column is merged color separation captured with the fluorescence microscope. (A) Numerous HEVs with strong CCL21 mRNA expression are detected in mouse mesenteric lymph node (MLN). HEVs (arrows) in left column are enlarged in the middle and right columns. Because of masking of epitopes by large depositions of biotinyl tyramide/Fast Red substrate at the site of CCL21mRNA, only thin streaks of PNAd expression are seen in these mouse HEVs (right), whereas more extensive PNAd expression is seen in the sense control (inset). (B) Extensive expression of CCL21 mRNA is seen in the T-cell zone of human MLN but is not detectable within HEVs. Negative HEVs (arrows) in left column are enlarged in the middle and right columns. (C) Extensive CCL21 mRNA expression is also seen outside HEVs in the T-cell areas in human tonsil. Negative HEVs (arrows) in the left column are enlarged in the middle column. No CCL21 mRNA is detected in the numerous MECA-79⁺ HEVs in the T-cell area (right). (D) Human PP with extensive CCL21 mRNA expression in T-cell areas. Vascular structures that express CCL21 mRNA are seen, but those with HEV morphology (arrow) are negative. CCL21 mRNA⁺ vascular structure with flat endothelium consistent with lymphatic vessel (lower right; arrowhead) is seen close to a MECA-79⁺ HEV (arrow), which is negative. Bar represents 100 μm except in the lower right panel, where it represents 50 μm.