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Fig. 6 | BMC Biology

Fig. 6

From: Differential DARC/ACKR1 expression distinguishes venular from non-venular endothelial cells in murine tissues

Fig. 6

Intravital microscopy (IVM) of leukocyte adhesion in DARC+ venules. a Schematic representation of IVM experiment. b Alexa Fluor 488 labeled anti-mouse DARC MAb staining of microvessels in subiliac lymph node. Branching order designation of intranodal venular segments was performed as in ref. [5]. Left: micrograph at the LN hilus showing a large DARC+ CV (order I) that drains venous blood into a DARC– extra-lymphoid vein (ELV); 20× objective. Right: Representative intranodal venular tree with branching orders identified in Roman numerals. c DARC expression in BM intermediate venules (IV), collecting venules (CV), post-sinusoids venules (PSV), and sinusoids (s). Microvascular segment designation was as in [31]; 10× objective (n = 3 experiments). d Intravital fluorescent micrographs of murine ear skin after injection of fluorescent leukocytes. Images were taken during an initial control period (left), after injection of Alexa Fluor 488 labeled anti-mouse DARC MAb (middle) and after subsequent injection of FITC-dextran, which delineates the intravascular compartment in all microvessels. Leukocytes rolling in DARC+ venules are identified by arrowheads. Arteriole (art.), venule (ven.); 20× objective (n = 3 experiments). See Additional file 10: movie 1. e Leukocyte rolling in DARC+ venules in cremaster muscle; 10× objective. Arrowhead in left panel indicates a non-adherent leukocyte captured while passing through an arteriole. See Additional file 11: movie 2. f Dashed arrowhead indicates pronounced DARC expression at the junction between adjacent ECs in cremaster muscle; 40× objective. g Rolling fractions (percent of rolling cells in the total flux of cells passing a microvessel) of calcein-labeled neutrophils in ear skin microvessels was assessed; n = 3 mice, data points from same animal are highlighted in same color (black, red or green). Error bars show mean ± SEM. ***P < 0.001. Supporting data values for this analysis are included in Additional file 2

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