The Prox1-Cre-tdTomato mouse facilitates FACS sorting of LECs.

<p>An ear single cell suspension was analyzed with a BD Fortessa flow cytometer. The tdTomato+ population contained 60% of CD45- CD31+ podoplanin+ LEC and 40% of CD45- CD31- cells. The sorting of tdTomato+ CD31+ cells allowed the isolation of a pure LEC population. Data are representative of 4 transgenic animals analyzed from 2 independent litters.</p

Comparison of the Prox1-Cre-tdTomato mouse with the Prox1-Cre-tdRFP mouse.

<p><b>A</b> Intravital confocal microscopy revealed brighter lymphatic vessels in the Prox1-Cre-tdTomato mouse than in the Prox1-Cre-tdRFP mouse (images acquired applying the same laser power and settings). <b>B</b> LVs can be visualized in Prox1-Cre-tdRFP mice only by applying a higher laser power. <b>C</b> Intravital multiphoton microscopy allowed the imaging of deep lymphatic collectors (arrowheads indicate valves), which were better visualized in Prox1-Cre-tdTomato mice. <b>D</b> Orthogonal analysis showed better visualization of the lymphatic vessel lumen in Prox1-Cre-tdTomato mice than in Prox1-mOrange mice, suggesting diffuse dye distribution in LECs. Scale bars: 100 μm.</p

TdTomato is expressed in the skin upon crossing of the reporter mice with a K5-Cre-ERT2 line.

<p><b>A</b> Schematic representation of the breeding to K5-Cre-ERT2 and of 4-hydroxytamoxifen (4-OHT) applications on the shaved back skin (1 mg in ethanol for five consecutive days). <b>B</b> Prior (day 0) and after 4-OHT application (day 7), mice where imaged with the IVIS spectrum (excitation: 570 nm; emission: 620nm; exposure time: 10 sec; binning: HR4. Color scale min = 2600 counts, max = 13000 counts). TdTomato was expressed in the skin of double positive, treated animals. Dashed line indicates the 4-OHT treated shaved back skin.</p

Generation of the tdTomato reporter mouse.

<p><b>A</b> Schematic representation of the LSL-tdTomato construct used for pronuclear microinjection. <b>B</b> HEK293 cells were transiently transfected with the construct with or without the STOP cassette. TdTomato fluorescence was analyzed 48 hrs post transfection with an inverted microscope and was visible only upon excision of the STOP cassette. <b>C</b> PCR genotyping of genomic DNA extracted from ear biopsies identified founder animals (f.l. founder line) positive for the transgene (tdTomato; 191 bp amplicon). A control gene (IL-2; 350 bp amplicon) was used for genomic DNA quality. <b>D</b> Transgene relative copy number was estimated by real-time PCR of genomic DNA. Primers specific for the transgene and for a control gene were used and the delta Ct was calculated. Founder 2 carried the least amount of copies, founder 4 the highest and founder 26 an intermediate number.</p

The Prox1-Cre-tdTomato mouse is a valid tool for intravital microscopy of DC migration.

<p><b>A</b> Maximum intensity projection of a z-stack of a mouse ear imaged with the IVM settings. The tdTomato positive LVs were clearly visible. Scale bar: 70 μm. <b>B</b> YFP-DCs were injected into the ear and imaged for 1 hour in an inverted confocal microscope. The image corresponds to the maximum intensity projection of single frame z-stack where the migratory tracks of DCs are shown. Scale bar: 70 μm. <b>C</b> Orthogonal view of the entry of a DC into the LV is visualized over time. Four representative time points from a 1-hour video are shown. Scale bar: 20 μm.</p

TdTomato expression co-localizes with lymphatic markers in different organs.

<p><b>A</b> Whole mount preparations of ear samples showed co-localization of the lymphatic marker LYVE-1 with tdTomato (detected with an anti-RFP antibody). Dashed line indicates a lymphatic collector vessel (LYVE-1 low, tdTomato high). <b>B</b> Whole mount preparations of ear samples showed co-localization of the lymphatic marker Prox1 with tdTomato. Upper panels show lymphatic collectors, middle panels show lymphatic capillaries and lower panels show a magnification of the area indicated in the middle panels. <b>C</b> FACS single cell analyses revealed tdTomato expression in the LEC population (CD45- CD31+ podoplanin+), but not in the BEC (CD45- CD31+ podoplanin-) or in the leukocyte (CD45+) populations. <b>D</b> Ultramicroscopic analysis of an immunostained and optically cleared lymph node showed colocalization of LYVE-1 and tdTomato. Maximum projection of a 260 μm z-stack. <b>E</b> Whole mount preparation of a lymph node sample showed the presence of LYVE-1+RFP+ lymphatic endothelium in the subcapsular sinus. LYVE-1+RFP- single cells are most likely macrophages. Data are representative of 4 transgenic animals analyzed from 2 independent litters. Scale bars: 100 μm.</p