Live immunostaining detects only extracellular basement membrane-bound CCL21, while staining on fixed tissue reveals intracellular as well as extracellular CCL21.

<p>(<b>a–b</b>) In the live ear dermis CCL21 accumulated in patches (arrowheads) and along continuous lymphatic segments (arrows), and co-localized with (<b>a</b>) perlecan and (<b>b</b>) collagen IV, both basement membrane components of the collecting lymphatic vessels. (<b>c</b>) Occasionally observed CCL21-positive (green) initial lymphatic vessels (iLy) stained more weakly for collagen IV (red). (<b>d</b>) Extracellular CCL21 deposits (green) were seen around lymphatic vessels in the exposed ear skin did not correlate with injured blood vessel, as determined by i.v. injection of TRITC-dextran (cyan) that leaked from areas of injured vessels (arrowheads). The tissue was pre-stained for perlecan and CCL21 before dextran injection. Scale bars in a, b and d (left), 400 µm; c and d (right), 100 µm.</p

Antibodies against collagen IV (basement membrane) served to identify most tissue structures in the dermis during intravital imaging.

<p>(<b>a</b>) Confocal imaging of the whole-mount stained tissue for the markers indicated. (<b>b</b>) Intravital immunofluorescence (IF) on the dorsal ear using collagen IV reveals the same structures except arterial smooth muscle cells (SMCs) and most capillary pericytes, with insets (i)–(iv) corresponding to zoomed areas shown at left by indicated boxes. Lymphatic collectors (Ly), initial lymphatics (iLy), veins (Ve), arteries (Ar), capillaries (c), nerve fibers (n), adipocytes (f), muscle fibers (m), SMC (p), collagen impressions of SMC (pi). Arrows indicate bicuspid valves of veins and collecting lymphatics; arrowheads indicate asymmetric valves of the pre-collecting lymphatics. Detection reagents: (a), collagen IV-streptavidin Alexa 700, VE-cadherin-Alexa 488, and αSMA-Alexa 594; (b), collagen IV-streptavidin Alexa 647. Scale bars in a, 20 µm; b, 500 µm (left) and 50 µm (insets).</p

Intravital immunofluorescence identifies extracellular stores of CCL21.

<p>(<b>a</b>) Immunostaining in the live ear (left) reveals only extracellular CCL21, which is extensively deposited on fragments of basement membrane-rich collecting lymphatics. In contrast, after fixing and re-staining for CCL21 of the same tissue, wide-field epifluorescence stereomicroscopy (middle) and confocal microscopy (right), detected intracellular stores of CCL21. Arrowhead points to CCL21 deposit on collectors basal membrane that were detected with intravital IF; arrows point to example location where peri-nuclear (intracellular) CCL21 signal develops only after the same tissue is fixed and permeabilized before re-staining. (b–e) Anti-CCL21 antibody does not substantially block activity of CCL21 deposits. (b) Extrinsic mouse CCL21 (50 µg/ml) was applied topically on a small delineated portion of the exposed ear dermis, and was observed bound to extracellular matrix and cell surfaces, and was detected with an antibody against intrinsic CCL21. (c) Immunostaining with anti-CCL21 antibodies did not block signaling that drove the directional migration of bone marrow-derived EGFP leukocytes towards CCL21 that had been pre-adsorbed on the tissue matrix. (d) When exogenous CCL21 was applied to the ear dermis at only 1 µg/ml, CCL21 could not be detected in the exposed part of the extracellular matrix, and at this 50-fold lower concentration, directed migration of leukocytes towards the region was absent. Instead bone marrow-derived EGFP leukocytes migrated towards the direction of the intrinsic CCL21 deposits (e). Arrows in b and d point to the edge of the topically applied CCL21 zone. Arrowheads in c and e point the direction of cell movement. Detection reagents in a: collagen IV-streptavidin Alexa 647, CCL21-Alexa 488 (stereomicroscope), CCL21-Alexa 594 (confocal). In: b–e: collagen IV-streptavidin Alexa 647, CCL21-Alexa 488. Scale bars in a, 50 µm; b and d, 500 µm; c and e, 100 µm.</p

Intravital immunofluorescence reveals different migratory behavior between leukocyte subsets in the skin.

<p>(<b>a</b>) Bone marrow-derived dendritic cells (BMDCs), isolated from the EGFP mouse and matured with LPS, were overlaid onto the Lyve1-α-rabbit Alexa 488 (red) stained ear dermis, and after 30 min the tissue was imaged for 3 hours. BMDCs were tracked (example tracks shown in white); (i) and (ii) show sequential images of two indicated areas where BMDCs (arrows, with yellow tracks shown at right) were observed entering Lyve1⁺ initial lymphatic vessels. (<b>b</b>) After staining for collagen IV (streptavidin-Alexa Pacific Blue, red) and CD45 (Alexa 596, green), the ear skin was stimulated with CVF-treated serum (anaphylatoxins) and imaged. Left, arrowhead points to pericyte imprint, indicative of collecting lymphatic vessels. Migrating CD45⁺ cells, most likely dermal dendritic cells (DDCs), were tracked (white). Insets (i) and (ii) show DDCs (arrows) entering collecting lymphatic vessels, with their migratory tracks (yellow) shown at right. (<b>c</b>) <i>Ex vivo</i> isolated peritoneal macrophages were stimulated with LPS and stained for CD11b-Alexa 594 before overlaying onto PECAM-1-Alexa 488 stained dermis. After 30 min, the tissue was imaged for 2 hours. Inset area (white box, left; sequential images, right) highlights the slow speed of migration of these cells compared to the DCs. (<b>d</b>) Quantification of cell migration speeds in (a)–(c). BMDCs and DDCs migrated with roughly the same speeds while macrophages remained immotile; since BMDCs were 5 times larger than intrinsic DDCs, their relative speed of migration (speed divided by cell diameter) was 5 times slower (p<0.0001). Scale bars, 50 µm.</p

Leukocyte migration and extravasation events in anaphylatoxin-stimulated tissue can be imaged with intravital immunofluorescence (IF).

<p>(<b>a</b>) Sequential images of the ear dermis of a wild-type mouse, stained for collagen IV (red) and Lyve1 (white) and transfused with blood from an EGFP mouse. Tissue stimulated with cobra venom factor (CVF, an anaphylatoxin)-treated serum caused leukocytes (arrows) to roll within a venule (arrowhead), transmigrate across venules, and migrate within the tissue. (<b>b</b>) Sequential images show the extent to which stimulation with serum and CVF (anaphylatoxins, bottom row) drives massive extravasation of leukocytes from blood vessels, compared to that under control conditions (top row). (<b>c</b>) Quantification of tracked leukocytes does not only show 15 times increase in leukocytes extravasation after CVF treatment, but also that their migration speeds were significantly faster. (<b>d</b>) Sequential images from an EGFP-chimeric mouse showing number of leukocytes entering CCL21-zone with a single leukocyte potentially entering pre-lymphatic collecting vessels and then stopping at the basement membrane for 8 minutes (6 to 14 min), squeezing its cytoplasm at the level of the CCL21-positive pre-collecting lymphatic vessel (9 to 14 min), and subsequently gaining the rounded-cell morphology (17 min). Arrowheads point to front and tail of the potentially transmigrating cell. Arrows point to cells that are crawling over basement membrane of the collecting lymphatic vessel. Tissue was stimulated with anaphylatoxins from CVF treated serum. Squares in image at 0 min represent example regions of interests (ROIs) that were placed onto either a CCL21-positive zone (i) or in a control zone (ii, dashed square) and used to calculate the relative density of cells entering CCL21 and control zones. (<b>e</b>) Leukocytes preferentially migrated towards intrinsic CCL21 immunolabeled depots. Cross correlation intensity from 6 pairs of sequential images increases in CCL21-ROIs and it is significantly different from control-ROIs, with p-value declining from 0.13 (15 min), 0.03 (30 min), to 0.008 (45 min). Error bars represent standard deviation. In: b–e: collagen IV-streptavidin Alexa 647, CCL21-Alexa 488. A–c, blood from EGFP mouse was transfused to WT after the tissue was stained with antibodies as indicated; d and e, bone marrow from EGFP mouse was transplanted to WT mouse two months before the experiment. Detection reagents: collagen IV-streptavidin-Pacific Blue, CCL21-Alexa 594, white-Lyve1-Alexa 488. Scale bars, 100 µm.</p