Lymphatic vessels are apparent before obvious TLOs. (A) H&E, DAPI, B220, and LYVE-1 staining in the kidney of a RIPLTα mouse (40× objective; Zeiss Axioscope). LYVE-1 positive lymphatic vessels (red) are within and around B cell regions (green) of the TLO. (B) In situ hybridization reveals LTα expression (dark purple) in the convoluted tubules of a RIPLTαTetOn mouse kidney after 1 month of doxycycline feeding (20× objective; Zeiss Axioscope). (C) Merge of bright field staining for hematoxylin and dark field for LYVE-1 positive vessels (red) reveals lymphatic vessels within kidney parenchyma after 2 months of doxycycline feeding before apparent extensive leukocytic infiltration. At 6 months, TLOs are apparent in the kidney with more obvious lymphatic vessels Cy-2 green LYVE-1 staining has been digitally colorized red to enhance contrast against hematoxylin (original magnification ×40; Zeiss Axioscope).