Intravital Immunofluorescence for Visualizing the Microcirculatory and Immune Microenvironments in the Mouse Ear Dermis

Intravital imaging of inflammatory and remodeling processes has been an important area of research and has also motivated the creation of numerous transgenic reporter mouse models that express fluorescent proteins. Our paper [1] describes a new in vivo imaging technique, which design was based on innovative concept of using immunostaining for live cells and tissue structures on surgically exposed mouse dermis without causing harmful immunotoxic effect. The surgical procedure itself was safe to the dermis vasculature as it relies only on separation of two skin layers in the ear that are independently innervated, autonomously fed by blood and drained by separated lymphatic circulations.


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To date, immunostaining in live tissue is not typically used due to formation of immune complexes leading to high staining background and immunotoxicity. We overcame these major problems by pre-blocking Fc receptors on tissue macrophages, thus "blinding” these cells to subsequent indirect strong immunostaining. Since the labeling was extracellular, we control strong phototoxicity and bleaching by immersion of tissue in natural antioxidant, the ascorbic acid (Figure 1).

Fig. 1: Intravital immunostaining and live imaging of dermis is possible after Fcγ receptor blocking and inhibition of phototoxicity. (a) A small posterior region of the ear was first exposed (dotted box), stained for Lyve1 and detected with a secondary antibody. Subsequently, the entire ear was exposed, blocked with irrelevant immunocomplexes for 15 min, and probed as before. Top inset: without Fcγ receptor blocking, high background results from immunocomplexes that form between primary and secondary antibodies and bind Fcγ receptors on tissue-resident immune cells. Bottom inset: this background is largely gone with pre-blocking of Fcγ receptors. (b) Photobleaching was minimized by adding ascorbate, an antioxidant, to Ringer’s buffer that bathed the ear tissue during imaging. Tissue was stained with a biotinylated antibody against collagen IV and streptavidin-Alexa 647 (red), and then constantly imaged for 300 s in either unmodified Ringer’s buffer (upper panel) or ascorbate-Ringer (lower panel); to demonstrate the extent of photobleaching, the brightest 25 % of pixel intensity values are shown in green (“high”). Note that the protecting effect of the ascorbate can be underestimated as the single exposure time for Ringer (control) was 227 ms while the exposure time for ascorbate was 427 seconds. (c) Quantification of fluorescence decay over time (30 % in Ringer’s vs. 5 % in ascorbate-Ringer), normalized to the initial fluorescence, averaged from 3 imaging fields; standard errors are shown by the dotted lines and p < 0.0001 between the two lines. Scale bars in a, 200 μm; b, 50 μm [1].

We also implemented the method for specific imaging of the tissue microenvironment with blood and lymphatic vessels, pericytes, nerves, muscle and adipocytes by staining for the basement membrane marker, e.g. collagen type IV, the basement membrane protein, instead of imaging second harmonic signal from non-structural fibrillar collagens. We present detailed design of the experimental setup and details for imaging a range of important physiological events over 12 hours, including leukocyte trafficking between blood and lymphatic vessels. We also implement this technique for imaging cancer cell invasion and metastasis, and immune cells homing to tumors (not shown). Such events can be imaged with simultaneously recording local physiological parameters such as blood vessel permeability and lymphatic drainage. Intravital immunofluorescence importantly also allows extracellular chemokine stores to be correlated with cell migration events.

Because the ear dermis is virtually two-dimensional, we could readily collect such data using fluorescence stereomicroscopy (Figure 2), instead of slower and more expensive confocal or multiphoton microscopy. In Leica fluorescent stereomicroscope main optics is embedded within microscope stand, which results in linear magnification from 16 to 320 times. In this system the main function of the lens is to collect maximum amount of light at the same time allowing the lens working distance to be relatively large (for 2x lens it is 2 cm working distance). This, in combination with fully automated stage, results in a comfort of manipulation and imaging allowing routinely more than one ear to be imaged in one experiment.

Fig. 2: Blood circulation in the surgically exposed dorsal ear dermis is functional. (a, b) Bright-field images showing the blood circulation (a) immediately after surgery and (b) after 20 min in the same ear, where circulation appears fully restored in all major blood vessels. (c) Lyve1 staining (in a different ear) shows the lymphatic vessel network, where the anterior part of the ear cannot be imaged with wide-field microscopy due to underlying adipose tissue of the hypodermis. (d) Functional drainage of TRITC-dextran by lymphatic vessels. TRITC-dextran was injected intradermally in the top of the dorsal skin that was stained for collagen IV. (e) Functionality of blood vessels in the exposed dermis as visualized with intravascular TRITC-dextran (red) showing damaged vessels (arrowheads) that leak dextran into the interstitial space. (f) Blood vessel permeability was assessed in the collagen IV-stained ear tissue after i.v. injection of 155 kDa TRITC-dextran. (f) Basal level of leakage over 30 min (Ctrl; top) and in the same tissue after addition of 1 μg/ml VEGF-A (bottom). (g) Relative increase in fluorescence intensity in the extravascular space as a function of time, showing quantification of vascular leakage. Shown are averages (triangles and squares) and standard errors (dotted lines) from 10 imaging fields. p < 0.0001 between the two lines. The following images come from the same experiment (separated by semi-colon): Fig. 1a and 1b; Fig. 1c; Fig. 1d; Fig. 1e; Fig. 1f. Scale bars in a, b, and c, 2 mm; d, f, 100 μm; e, 500 μm [1].

In our publication we compare our technique to state-of-the-art standards in various fields of intravital imaging. Consequently, we observed described earlier blood vessels extravasation and lymphatic intravasation events of various types of leukocyte with unique visualization of immune cells entering collecting instead of initial lymphatic vessels. We also identified the major modes of metastatic cells migration from the orthotopically implanted melanoma (not shown). We showed that in vivo, CCL21 is able to form strong, discontinuous deposits on collecting but only infrequently on initial lymphatics. This is intriguing, as collecting vessels have up to date been ignored as potential portals of entry for immune cells. In fact, we showed that dermal dendritic cells and granulocytes could enter collecting lymphatic vessels, an observation that not only adds physiological importance to the discovery of the discontinuous CCL21 patches located on collectors, but which could also switch the paradigm in lymphatic and CCL21 biology as intravasation into this type of vessels had not been observed before. How important this discovery could be is the fact that very similar to our but complementing observation of weak and continuous CCL21 haptotactic gradient along initial lymphatics was published recently in Science by Weber et al. [3]. Our paper complements the research of Weber et al., because we provided a potential explanation for relatively infrequent localization of strong CCL21 deposits on initial lymphatics as compared to collecting ones. We suspect that the tick basement membrane of lymphatic collectors with abundant presence of perlecan and agrin, the first tissue receptors encountered by lymphatic-derived CCL21, is better in capturing heparan-binding chemokine than initial lymphatics that are weakly supported by the basement membrane (Figure 3).

Fig. 3: Live immunostaining detects only extracellular basement membrane-bound CCL21, while staining on fixed tissue reveals intracellular as well as extracellular CCL21. (a–b) In the live ear dermis CCL21 accumulated in patches (arrowheads) and along continuous lymphatic segments (arrows), and co-localized with (a) perlecan and (b) collagen IV, both basement membrane components of the collecting lymphatic vessels. (c) Occasionally observed CCL21-positive (green) initial lymphatic vessels (iLy) stained more weakly for collagen IV (red). (d) Extracellular CCL21 deposits (green) seen around lymphatic vessels in the exposed ear skin did not correlate with areas of blood vessel injury, as determined by i.v. TRITC-dextran (cyan) seen leaking from areas of injured vessels (arrowheads) after the tissue was stained for perlecan and CCL21. Scale bars in a, b and d (left), 400 μm; c and d (right), 100 μm [1].


This method should be interesting and useful to the scientific community, particularly in the areas of microcirculation, immunology and tumor microenvironment including studies on tumor angiogenesis and anti-angiogenic therapy. It has some advantages over other intravital imaging techniques: (i) Intravital fluorescence imaging has been used in microcirculation studies, e.g., to track solute leakage from blood or into lymphatics, but has not been combined with immunostaining. (ii) The use of genetically modified reporter mice allows for specific cell types to be imaged, but requires their availability (or substantial effort to create new ones) and limits the number of interacting cell types that can be studied. (iii) Extracellular matrix can be imaged in vivo using second harmonic generation, but this is only limited to fibrous collagens, leaving large number of important matrices like basement membranes, fibronectins or growth factor and chemokine binding glycosaminoglycans out of reach for current research. Our method overcomes all of these limitations, and furthermore allows standard imaging techniques to be incorporated and further combined with immunostaining for other cell types, tissue structures, chemokine deposits, or extracellular matrix proteins while simultaneously tracking blood or lymphatic flows. Finally, this method received attention even before it was published. The image showing melanoma cells invading live lymphatic collector vessels was chosen as a cover picture of the Nature review article “Tracking metastasis and tricking cancer” [2].

In summary, this intravital immunofluorescence bridges real-time, local measurements of physiological function with molecular imaging of complex cellular events in skin. Furthermore, this method has far greater potential, as it can be easily applied to e.g. study post-developmental mechanisms of blood and lymph-angiogenesis, live imaging of transplant rejection, or visualizing the early phases of skin infection by various pathogenic agents. For example we are finishing the manuscript where by using this intravital method we identified the mechanism of filaria infection and entry into lymphatics. We are also analyzing the effect of the heterogeneity of the tumor extracellular matrix on metastasis and angiogenesis. With its flexibility and high-throughput potential this intravital technique can revolutionize multiple fields of biology.

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