We thank Thomas Graf for providing us with the LysM-eGFP mice.

All experiments dealing with live animals were conducted in accordance to all relevant animal use and care guidelines and regulations.
1. Choice of Fluorescent Reporter Mice
<ol>
	<li>Use 6- to 12-week-old LysM-eGFP11 mice (no preference for either males or females). 
	Note: The use of various cell-specific fluorescent reporter mice enables the visualization of different immune cells in vivo. In this strain, circulating neutrophils (GFPhi cells), circulating monocytes (GFPlo cells), and dermal macrophages (GFPlo cells) can be visualized. With the imaging parameters used, only the bright signals from GFP-positive neutrophils will be detected. 
	Note: A list of immune-cell-specific fluorescent reporter mouse strains suitable for this type of skin imaging study can be found in Reference 8. 
	Note: It is highly recommended that albino mice be used for imaging, as pigmented mice are more prone to photodamage. This is because the pigmented ear skin is much more sensitive to laser-induced speckling (indicative of tissue burning). As a result, neutrophil recruitment and accumulation may be observed even during the steady state8,12.</li>
	<li>Keep the mice in specific pathogen-free (SPF) conditions with 12-h light-dark cycles.</li>
</ol>
2. Mouse Anesthesia
<ol>
	<li>Anesthetize the mouse with an intra-peritoneal injection of ketamine-xylazine (8 &#181;L g-1 bodyweight), composed of a mixture of 15 mg mL-1 ketamine and 1 mg mL-1 xylazine dissolved in sterile water.</li>
	<li>Place the mouse on a heating pad to maintain its body temperature at 37 &#176;C throughout the preparation procedure. Check for sufficient anesthesia by observing an absence of a toe pinch reflex. 
	Note: After the first hour, subsequent quarter doses of anesthetic will need to be administered subcutaneously and will last for approximately 0.5 h each. The twitching of whiskers or the tail may also indicate that the anesthesia is wearing off and that a top-up is required.</li>
	<li>Use ophthalmic lubricant on the eyes to prevent dryness while under anesthesia.</li>
</ol>
3. Depilation
<ol>
	<li>Carefully apply depilatory cream to the upper two-thirds of the dorsal mouse ear using cotton-tip applicators.</li>
	<li>Wait for 2 - 3 min before removing the cream using wet cotton-tip applicators in a thorough but gentle manner. 
	Note: Do not allow hair removal cream to stay on the mouse ear for too long, as it may induce inflammation13,14.</li>
</ol>
4. Induction of Ischemia and Reperfusion Injury
<ol>
	<li>Use gold-plated, N42-grade neodymium magnets, 12 mm diameter x 2 mm thick, and with a Gauss rating of approximately 3,000 to induce ischemia in the mouse ear skin. 
	Note: In this case, the dimpled face of the magnets denotes its north pole.</li>
	<li>Slot the magnets into their individual plastic guides. 
	Note: The plastic guide serves to ease the placement and separation of the high-strength magnets. Due to their strong magnetic force yet low resistance to breakage, do not place individual magnets in close proximity to each other or to other metals. Breakage and splintering may occur if they pull towards each other.</li>
	<li>Position the first (dorsal) magnet such that only the edge is in contact with the second (ventral) magnet (Figure 3a) 
	Note: This prevents the magnets from snapping together before they have been properly positioned.</li>
	<li>Position both magnets such that the ventral magnet lies flat on the ear (Figure 3a). 
	Note: Before ischemia is induced, ensure that the mouse is kept at 37 &#176;C and that sufficient anesthesia is maintained (see step 2.2).</li>
	<li>Once ready, carefully let the magnets come together (Figure 3a). 
	Note: For imaging purposes, clamp only half of the ear so that an IR and non-IR region can be observed.</li>
	<li>After 1.5 h of ischemia, remove the magnets by twisting the magnets away from each other using the plastic guides, allowing reperfusion to take place. 
	Note: Care must be taken to prevent the ears from creasing when the magnets are placed. Incomplete ischemia is evident if the macroscopically visible major blood vessels re-perfuse immediately (i.e., blood fills the vessels immediately) after the magnets are removed. Although reperfusion does not occur immediately after the removal of magnets, blood vessel occlusion is only transient. As such, it is imperative to prepare the mouse ear for imaging as swiftly as possible.</li>
</ol>
5. Injection of Blood Vessel Labeling Agents
<ol>
	<li>Immediately after magnet removal, administer intravenously (via retro-orbital or tail vein injection) Evans blue (10 mg mL-1 in PBS or saline; 1 &#181;L g-1 bodyweight) or another blood vessel labeling agent of choice. 
	Note: Before injection, ensure that sufficient anesthesia is still maintained by performing gentle toe pinching.</li>
</ol>
6. Placement of the Ear on the Imaging Platform
<ol>
	<li>Cut 2 pieces of masking tape 1.5 cm in length and 1.8 cm in width.</li>
	<li>Allow the adhesive sides to stick together whilst leaving about 1 mm of adhesive along its width.</li>
	<li>Cut the masking tape in two, lengthwise, to accommodate its placement within the slit on the ear platform.</li>
	<li>Insert this masking tape about halfway through the slit, such that the adhesive side faces up.</li>
	<li>Position the mouse on the heating pad, such that the ear to be imaged is next to the masking tape strip.</li>
	<li>By using two PBS-moistened cotton-tip applicators, gently press the ear against the adhesive strip.</li>
	<li>Using the strip as a guide, bring the mouse ear through the slit while simultaneously adjusting the mouse closer towards the stage.</li>
	<li>To remove the sticky tape, first add a drop of PBS to reduce the adhesiveness of the tape.</li>
	<li>Separate the mouse ear from the masking tape as gently as possible using a fine paintbrush.</li>
	<li>Flatten the ear against the ear platform by gently rolling a moist cotton-tip applicator over the ear.</li>
	<li>Put a drop of PBS underneath the coverslip (which is kept in position on the coverslip holder using grease; Figure 2) and gently place it over the ear. Top up with more PBS if necessary. 
	Note: The coverslip holder increases the stability during imaging.</li>
	<li>Insert the rectal temperature probe and connect the wires to the heating system according to manufacturer&#39;s instructions. 
	Note: Set the temperature of the body heating pad to 37 &#176;C and the ear stage platform to 35 &#176;C.</li>
</ol>
7. Multiphoton Microscope Setup and Imaging Parameters
Note: This protocol uses a single beam, multiphoton microscope with a tunable (680 - 1,080 nm) Ti:Sa laser (3.3 W at 800 nm; pulse length of 140 fs; 80 MHz repetition rate) with a 20X water objective (NA = 1.0) for intravital imaging studies.
<ol>
	<li>Open the imaging software.</li>
	<li>Align the laser according to the manufacturer&#39;s instructions.</li>
	<li>Adjust the excitation wavelength to 950 nm. 
	Note: GFP and Evans blue can be simultaneously excited at 950 nm.</li>
	<li>To preview, use the following settings: 500 &#181;m2 scanfield, 505 x 505 pixel resolution, and a scan frequency of 800 Hz in a single line scan. Click &#34;Preview.&#34;</li>
	<li>Toggle the attenuator (laser) power. Ensure that all essential signals are picked up without exposing the imaging field to excessive amounts of laser power, which may induce heat damage. 
	Note: Start at a low attenuator power and increase if the signal is dim. As neutrophils will be absent from the interstitium at early time points post-reperfusion, macrophages may be used as a gauge to determine the minimal power required, as the former are dimmer than neutrophils. 
	Note: If this step is to be done for the first time, adjust the settings in the non-ischemic zone, where intact vascular integrity is expected and will facilitate the setting of the attenuator power. In subsequent experiments, these settings do not require much modification unless the laser power is unstable.</li>
	<li>Adjust the PMT settings. 
	Note: Check with the manufacturer for the maximal optimal gain voltage for the PMT. Setting the PMT voltage beyond the recommended threshold will result in a higher signal-to-noise ratio. The general recommendation is to set the PMT gain voltage to the recommended threshold and to increase the attenuator power as necessary should the signal be too dim.</li>
	<li>Select an imaging region that is in close proximity to the edge of the ischemia, characterized by massive Evans blue leakage.</li>
	<li>Collect GFP and Evans blue signals using 525/50 bandpass (BP) and 655/40 BP filters, respectively. For second harmonic generation (SHG) signals of collagen fibers within the dermal compartment, use a 475/42 BP filter.</li>
	<li>Create a folder to save the images in a format that is compatible for the available image analysis software.</li>
	<li>To acquire, use the following settings: 500 &#181;m2 scanfield, 505 x 505 pixel resolution, and a scan frequency 400 Hz in a single line scan.</li>
	<li>A 100-&#181;m z-stack with a step size of 4 &#181;m can be acquired repeatedly over time, at 1 min intervals, to monitor neutrophil infiltration. 
	Note: Especially for leukocytes (e.g., neutrophils) with a higher migratory velocity, an interval that is longer than 1 min may result in difficulties during cell tracking analysis. In this case, the user can either reduce the thickness of the acquisition stack or increase the scan frequency. 
	Note: While the required acquisition stack of the ischemic zone may appear smaller as a result of the compression (alternatively characterized by a higher SHG intensity), subsequent reperfusion will result in massive inflammation that causes the ear to swell. Significant drifts in the Z-direction are expected. As such, the acquisition of a large z-stack is necessary to accommodate the drift.</li>
	<li>Throughout the imaging, top up the PBS and water regularly to keep the ear moist and the objective lens immersed in water. 
	Note: A typical experiment usually lasts for 2 - 4 h. Depending on the experimental design, and coupled with a well-controlled anesthesia regime, extending the duration of imaging is possible.</li>
</ol>
8. Terminating the Experiment
<ol>
	<li>Euthanize the mouse by carbon dioxide asphyxiation according to the institution&#39;s Institutional Animal Care and Use Committee (IACUC) procedures. 
	Note: Always euthanize the mouse by approved methods as determined by the institution&#39;s rules, regulations, and guidelines. If repeated imaging is needed, keep the mouse on the heating pad until the anesthesia wears off. Once the mouse has regained sufficient consciousness and is mobile, return the mouse to its cage.</li>
</ol>
9. Image Analysis
Note: Data generated from the imaging experiment can be visualized by different software packages.
<ol>
	<li>Open the imaging analysis software.</li>
	<li>Under Surpass Mode, import the file (Open &#8594; select any file in the desired folder &#8594; Click &#34;open&#34;).</li>
	<li>Edit pseudo colors if default colors do not apply (Edit &#8594; Display adjustment &#8594; Under the channel tab, select the desired color from the color palette).</li>
	<li>Adjust the brightness, contrast, and background (Edit &#8594; Display Adjustment &#8594; Toggle Max and Min values).</li>
	<li>Ensure that the file dimensions are correct (Edit &#8594; Image properties &#8594; Under the geometry tab, check the voxel size. In this protocol, X = 0.99, Y = 0.99, and Z = 4). 
	Note: Derive X and Y by dividing the scanfield dimension by the pixel resolution. Z is the thickness between each slice. 
	Note: The output of these data sets can be presented as maximum projection movies. 
	Note: Tracking analysis is required to fully characterize the cellular activities and interactions in order to understand their function in vivo. Detailed instructions of cell tracking using the spot function available in the software can be found in the protocol in Reference 15. 
	Note: There are many ways of quantifying leukocyte migration, details of which can be found in the review article in Reference 16.</li>
</ol>

<ol><li>Black, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((National+Pressure+Ulcer+Advisory+Panel%27s+updated+pressure+ulcer+staging+system%5BTitle%5D)+AND+%22Adv+Skin+Wound+Care%22%5BJournal%5D)">National Pressure Ulcer Advisory Panel's updated pressure ulcer staging system.</a> Adv Skin Wound Care. 20, 269-274 (2007).</li>
<li>Kalogeris, T., Baines, C. P., Krenz, M., Korthuis, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+biology+of+ischemia%2Freperfusion+injury%5BTitle%5D)+AND+%22Int+Rev+Cell+Mol+Biol%22%5BJournal%5D)">Cell biology of ischemia/reperfusion injury.</a> Int Rev Cell Mol Biol. 298, 229-317 (2012).</li>
<li>Huebener, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+HMGB1%2FRAGE+axis+triggers+neutrophil-mediated+injury+amplification+following+necrosis%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">The HMGB1/RAGE axis triggers neutrophil-mediated injury amplification following necrosis.</a> J Clin Invest. 125, 539-550 (2015).</li>
<li>Wassermann, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+chronic+pressure+ulcer+model+in+the+nude+mouse%5BTitle%5D)+AND+%22Wound+Repair+Regen%22%5BJournal%5D)">A chronic pressure ulcer model in the nude mouse.</a> Wound Repair Regen. 17, 480-484 (2009).</li>
<li>Stadler, I., Zhang, R. Y., Oskoui, P., Whittaker, M. S., Lanzafame, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+simple%2C+noninvasive%2C+clinically+relevant+model+of+pressure+ulcers+in+the+mouse%5BTitle%5D)+AND+%22J+Invest+Surg%22%5BJournal%5D)">Development of a simple, noninvasive, clinically relevant model of pressure ulcers in the mouse.</a> J Invest Surg. 17, 221-227 (2004).</li>
<li>Tsuji, S., Ichioka, S., Sekiya, N., Nakatsuka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+ischemia-reperfusion+injury+in+a+microcirculatory+model+of+pressure+ulcers%5BTitle%5D)+AND+%22Wound+Repair+Regen%22%5BJournal%5D)">Analysis of ischemia-reperfusion injury in a microcirculatory model of pressure ulcers.</a> Wound Repair Regen. 13, 209-215 (2005).</li>
<li>Ng, L. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+the+neutrophil+response+to+sterile+tissue+injury+in+mouse+dermis+reveals+a+three-phase+cascade+of+events%5BTitle%5D)+AND+%22J+Invest+Dermatol%22%5BJournal%5D)">Visualizing the neutrophil response to sterile tissue injury in mouse dermis reveals a three-phase cascade of events.</a> J Invest Dermatol. 131, 2058-2068 (2011).</li>
<li>Li, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravital+multiphoton+imaging+of+immune+responses+in+the+mouse+ear+skin%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Intravital multiphoton imaging of immune responses in the mouse ear skin.</a> Nat Protoc. 7, 221-234 (2012).</li>
<li>Tong, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+skin+immune+atlas%3A+three-dimensional+analysis+of+cutaneous+leukocyte+subsets+by+multiphoton+microscopy%5BTitle%5D)+AND+%22J+Invest+Dermatol%22%5BJournal%5D)">The skin immune atlas: three-dimensional analysis of cutaneous leukocyte subsets by multiphoton microscopy.</a> J Invest Dermatol. 135, 84-93 (2015).</li>
<li>Saito, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+loss+of+MCP-1+attenuates+cutaneous+ischemia-reperfusion+injury+in+a+mouse+model+of+pressure+ulcer%5BTitle%5D)+AND+%22J+Invest+Dermatol%22%5BJournal%5D)">The loss of MCP-1 attenuates cutaneous ischemia-reperfusion injury in a mouse model of pressure ulcer.</a> J Invest Dermatol. 128, 1838-1851 (2008).</li>
<li>Faust, N., Varas, F., Kelly, L. M., Heck, S., Graf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insertion+of+enhanced+green+fluorescent+protein+into+the+lysozyme+gene+creates+mice+with+green+fluorescent+granulocytes+and+macrophages%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages.</a> Blood. 96, 719-726 (2000).</li>
<li>Roediger, B., Ng, L. G., Smith, A. L., Fazekasde de St Groth, B., Weninger, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+dendritic+cell+migration+within+the+skin%5BTitle%5D)+AND+%22Histochem+Cell+Biol%22%5BJournal%5D)">Visualizing dendritic cell migration within the skin.</a> Histochem Cell Biol. 130, 1131-1146 (2008).</li>
<li>Kikushima, K., Kita, S., Higuchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+non-invasive+imaging+for+the+in+vivo+tracking+of+high-speed+vesicle+transport+in+mouse+neutrophils%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">A non-invasive imaging for the in vivo tracking of high-speed vesicle transport in mouse neutrophils.</a> Sci Rep. 3, 1913(2013).</li>
<li>Ng, L. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Migratory+dermal+dendritic+cells+act+as+rapid+sensors+of+protozoan+parasites%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">Migratory dermal dendritic cells act as rapid sensors of protozoan parasites.</a> PLoS Pathog. 4, e1000222(2008).</li>
<li>Soohoo, A. L., Bowersox, S. L., Puthenveedu, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+clathrin-mediated+endocytosis+of+G+protein-coupled+receptors+at+single-event+resolution+via+TIRF+microscopy%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Visualizing clathrin-mediated endocytosis of G protein-coupled receptors at single-event resolution via TIRF microscopy.</a> J Vis Exp. , e51805(2014).</li>
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</ol>

The authors have nothing to disclose.

Significance
IR is one of the leading causes of skin pressure ulcers. The early stages (I and II) of pressure ulcers describe the condition of the human skin (as compared to the underlying subcutaneous tissues and muscles). However, an understanding of the immunological etiology is still lacking. Here, we present a simple and robust IR model on mouse ear skin in order to address this gap. We simulate ischemia by clamping the mouse ear between two magnets and subsequently study the downstream immu...

Ischemia-reperfusion injury (IRI) occurs when there is a transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent re-oxygenation of the tissues (reperfusion). In the skin, ischemia-reperfusion (IR) is thought to be one of the contributing factors to the pathophysiology of pressure ulcers, where prolonged bed rest predisposes long-term hospital patients to injury. In these patients, both the skin and the underlying muscles are constantly exposed to weight pressure exerted over areas of bony prominence, resulting in localized injuries that, if left untreated, may become necrotic1.
The damages involved in an IRI are twofold. During ischemia, the occlusion of blood vessels leads to a drastic drop of oxygen delivery to the tissues. This results in a decrease of ATP and pH, which inactivates ATPases involved in cellular metabolism. In turn, cellular calcium levels spike, and stressed or damaged cells undergo apoptosis or necrosis2. The release of intracellular contents or damage associated molecular patterns (DAMP), like HMGB1, contributes to the inflammatory response3. The second insult occurs during reperfusion. Although oxygen and pH levels are restored during reperfusion, this results in the generation of reactive oxygen species (ROS), which leads to the oxidation of intracellular lipids, DNA, and proteins. Consequently, pro-inflammatory mediators are activated, which sets off a secondary inflammatory response that involves the recruitment of immune cells to the inflammatory site2. While the cascade of biochemical events leading up to the inflammatory response has been well described, the spatial and temporal regulation of the immune cell activities are not well understood.
Here, we describe a robust IR model on mouse ear skin using simple magnet clamping. Coupled with multiphoton intravital imaging (MP-IVM), we established a model to study the in vivo inflammatory responses that occur after reperfusion takes place. The rationale behind the development and use of this technique is to try to understand how both interstitial and infiltrating cells respond to IR in real time.
Existing models of IR using the clamping technique on the skin flank are highly invasive, as they require the surgical implantation of steel plates in the skin flank, making them less-than-ideal for immunological studies4. A similar non-invasive clamping technique has been described in the mouse skin flank5,6. However, because of the incorporation of the intravital imaging component in this method, we instead chose the ear skin as the targeted IR site, as it circumvents movements due to breathing and offers stability during imaging7,8. Moreover, leukocyte subsets that span the interstitium are identical between the ear skin and the skin flank, although the numbers and proportions may vary slightly9. Thus, the ear skin represents an ideal imaging site.
In addition, most data retrieved from these IRI models are limited to macroscopic evaluations (grading of ulcers) and microscopic analyses of endpoint inflammatory indicators10. Using this model, real-time visualization of the cellular response of neutrophils after reperfusion in the skin of a fluorescent reporter mouse is enabled. A previously published intravital ear imaging model is utilized8 with additional modifications (Figures 1, 2).

<table><tbody><tr><td colspan="4">&lt;strong&gt;Mice strains&lt;/strong&gt;</td></tr><tr><td>Lysozyme-GFP C57BL/6</td><td>Thomas Graf, Center for Genomic Regulation</td><td></td><td></td></tr><tr><td>C57BL/6-C2J</td><td>Jackson Laboratories</td><td>000058</td><td>To be crossed with Lysozyme-GFP to generate albino Lysozyme-GFP for skin imaging</td></tr><tr><td colspan="4">&lt;strong&gt;Reagents&lt;/strong&gt;</td></tr><tr><td>PBS</td><td></td><td></td><td></td></tr><tr><td>Viaflex 0.9% (wt/vol) saline</td><td>Baxter Healthcare</td><td>F8B1323</td><td></td></tr><tr><td>Ketamine (100 mg mL&lt;sup&gt;&amp;minus;1&lt;/sup&gt; ketamine hydrochloride</td><td>Parnell</td><td></td><td>Ketamine is a controlled drug and all relevant local regulations should be followed</td></tr><tr><td>Ilium Xylazil-20 (20&amp;nbsp;mg&amp;nbsp;mL&lt;sup&gt;&amp;minus;1&lt;/sup&gt; xylazine hydrochloride)</td><td>Troy Laboratories</td><td></td><td>Xylazil-20 is a controlled drug and all relevant local regulations should be followed.</td></tr><tr><td>Evans blue (10&amp;nbsp;mg&amp;nbsp;mL&lt;sup&gt;&amp;minus;1&lt;/sup&gt; in PBS or saline)</td><td>Sigma-Aldrich</td><td>46160</td><td></td></tr><tr><td>Ultrapurified water</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Insulin syringe with needle</td><td>BD</td><td>328838</td><td></td></tr><tr><td>Transfer pipettes</td><td>Biologix Research Company</td><td>30-0135</td><td></td></tr><tr><td>3 M paper masking tape</td><td>3M</td><td>2214</td><td></td></tr><tr><td>Deckglaser microscope cover glass (22 mm &amp;times; 32 mm)</td><td>Paul Marienfeld</td><td>101112</td><td></td></tr><tr><td>Curved splinter forceps</td><td>Aesculap, B. Braun Melsungen</td><td>BD312R</td><td></td></tr><tr><td>Veet hair removal cream</td><td>Reckitt Benckiser</td><td></td><td></td></tr><tr><td>Medical cotton-tipped applicators</td><td>Puritan Medical Products Company</td><td>806-WC</td><td></td></tr><tr><td>C-fold towels</td><td>Kimberly-Clark</td><td>20311</td><td></td></tr><tr><td>Kimwipes delicate task wipes</td><td>Kimtech Science</td><td>34155</td><td></td></tr><tr><td>Gold-plated, N42-grade neodymium magnets, 12 mm in diameter and 2 mm thick&amp;nbsp;</td><td>first4magnets</td><td>F656S</td><td></td></tr><tr><td>Plastic guide, 10 cm by 1.5 cm (polyvinyl chloride material)</td><td></td><td></td><td>fold in half lengthwise, bind with masking tape and slot magnet in</td></tr><tr><td>High vacuum grease</td><td>Dow Corning</td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Microscope&lt;/strong&gt;</td></tr><tr><td>TriM Scope II single-beam two-photon microscope</td><td>LaVision BioTec</td><td></td><td></td></tr><tr><td>Tunable (680&amp;ndash;1,080 nm) Coherent Chameleon Ultra II One Box Ti:sapphire laser (&amp;ge;3.3 W at 800 nm; pulse length of 140 fs, 80 MHz repetition rate)</td><td>Coherent</td><td></td><td></td></tr><tr><td>Water-dipping objectives (20&amp;times;, NA = 1.0)</td><td>Olympus</td><td>XLUMPLFLN20xW</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Miscroscope filter and mirror sets (for imaging GFP, SHG, Evans Blue)&lt;/strong&gt;</td></tr><tr><td>495 long-pass</td><td>Chroma</td><td>T495LPXR</td><td></td></tr><tr><td>560 lomg-pass</td><td>Chroma</td><td>T560LPXR</td><td></td></tr><tr><td>475/42 band-pass</td><td>Semrock</td><td>FF01-475/42-25</td><td></td></tr><tr><td>525/50 band-pass</td><td>Chroma</td><td>ET525/50m</td><td></td></tr><tr><td>655/40 band-pass</td><td>Chroma</td><td>NC028647</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Skin-imaging stage platform (refer to diagram for assembly)&lt;/strong&gt;</td></tr><tr><td>A metal base plate (126 mm &amp;times; 126 mm &amp;times; 1 mm)</td><td></td><td></td><td></td></tr><tr><td>A brass platform for the ear (79 mm &amp;times; 19 mm; 1 mm thickness at side, 0.5 mm thickness in the middle; &lt;strong&gt;Figure 1&lt;/strong&gt;) with slit (1.7 mm &amp;times; 1 mm; 1.5 mm away from long edge)</td><td></td><td></td><td></td></tr><tr><td>Two plastic blocks (10 mm in height)&amp;mdash;for heat insulation</td><td></td><td></td><td></td></tr><tr><td>Curved holder, for positioning the control thermistor on the ear platform</td><td></td><td></td><td></td></tr><tr><td>Interface cable CC-28 with DIN connector and thermistors, one for the temperature control and the other for the temperature monitor</td><td>(Warner Instruments (Harvard Apparatus)</td><td>640106</td><td>connect the interface cable to both resistive heater blocks set at 35 &amp;deg;C</td></tr><tr><td>Resistive heater blocks RH-2</td><td>(Warner Instruments (Harvard Apparatus)</td><td>640274</td><td>Resistive heater blocks can heat the brass ear platform up to over 100 &amp;deg;C within minutes. Ensure that the control thermistor has been properly secured in the holder in order to avoid overheating.</td></tr><tr><td>Temperature controller TC-344B for the ear platform</td><td>(Warner Instruments (Harvard Apparatus)</td><td>640101</td><td></td></tr><tr><td>Temperature controller TR-200 for mouse heating pad</td><td>Fine Science Tools</td><td>21052-00</td><td>Unit is no longer for sale. Ask manufacturer for alternatives</td></tr><tr><td>Power supply for TR-200</td><td>Fine Science Tools</td><td>21051-00</td><td>Unit is no longer for sale. Ask manufacturer for alternatives</td></tr><tr><td>Heating pad</td><td>Fine Science Tools</td><td>21060-00</td><td>Unit is no longer for sale. Ask manufacturer for alternatives.&amp;nbsp;</td></tr><tr><td>Animal rectal probe&amp;nbsp;</td><td>Fine Science Tools</td><td>21060-01</td><td>Unit is no longer for sale. Ask manufacturer for alternatives. After connecting the rectal probe and heating pad to the temperature controller TR-200, set the temperature to 37 &amp;deg;C</td></tr><tr><td colspan="4">&lt;strong&gt;Coverslip holder&lt;/strong&gt;</td></tr><tr><td>2 plastic rods, 1 cm in diameter, 10 cm in length</td><td></td><td></td><td></td></tr><tr><td>1 plastic adaptor with holes drilled to accommodate rods (refer to diagram)</td><td></td><td></td><td></td></tr><tr><td>3 plastic tightening screws for keeping plastic rods in place</td><td></td><td></td><td></td></tr><tr><td>1 metal plate, 6 cm x 2.5 cm, with a 2 cm square cut at 1 end, 2 mm edge away from short edge</td><td></td><td></td><td></td></tr><tr><td>1 pair of nut and bolt for attaching metal plate to plastic rod</td><td></td><td></td><td></td></tr><tr><td>1 acrylic base (4 cm x 5 cm x 1.5 cm) with magnet to hold coverslip holder on skin-imaging stage platform. 1 rod is permanently fixed onto base.</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Imaging analysis software&lt;/strong&gt;</td></tr><tr><td>Imaris v8.1.2</td><td>Bitplane</td><td></td><td></td></tr></tbody></table>

inducing ischemia-reperfusion injury in the mouse ear skin for intravital multiphoton imaging of immune responses

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent re-oxygenation of the tissues (reperfusion). In the skin, ischemia-reperfusion (IR) is the main contributing factor to the pathophysiology of pressure ulcers. While the cascade of events leading up to the inflammatory response has been well studied, the spatial and temporal responses of the different subsets of immune cells to an IR injury are not well understood. Existing models of IR using the clamping technique on the skin flank are highly invasive and unsuitable for studying immune responses to injury, while similar non-invasive magnet clamping studies in the skin flank are less-than-ideal for intravital imaging studies. In this protocol, we describe a robust model of non-invasive IR developed on mouse ear skin, where we aim to visualize in real-time the cellular response of immune cells after reperfusion via multiphoton intravital imaging (MP-IVM).

This protocol uses a custom-built ear skin imaging platform, as shown in Figure 1. Several features of this platform are specifically designed to facilitate imaging while maintaining physiological settings. Placing the ear on the heated brass platform not only maintains the ear at a physiological temperature of 35 &#176;C, but it also isolates the ear from inevitable movements due to breathing. The addition of a metal clip on the brass platform creates a gap to prevent th...

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Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

This protocol describes the induction of an ischemia-reperfusion (IR) model on mouse ear skin using magnet clamping. Using a custom-built intravital imaging model, we study in vivo inflammatory responses post-reperfusion. The rationale behind the development of this technique is to extend the understanding of how leukocytes respond to skin IR injury.

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent ...

inducing-ischemia-reperfusion-injury-mouse-ear-skin-for-intravital

Research

JoVE Journal

Medicine

11.2K Views.  Agency for Science, Technology and Research (A*STAR), Biopolis.  Dit protocol beschrijft de inductie van een ischemie-reperfusie (IR) model op de muis oor huid met behulp van magneet klemmen. Met behulp van een custom-built intravitale imaging model, bestuderen we in vivo ontstekingsreacties post-reperfusie. De reden voor de ontwikkeling van deze techniek is om te begrijpen hoe leukocyten reageren op huid IR schade verlengen. 

Video: Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

Bioengineering

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Immunology and Infection

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions between cellular and non-cellular blood components during thrombus formation, reliable studies of the physiology and pathophysiology of thrombosis can only be performed in vivo. Therefore, this article presents an ear model in hairless mice and focuses on the in vivo analysis of microcirculation, thrombus formation, and thrombus evolution. By using intravital fluorescence microscopy and the intravenous (iv) application of the respective fluorescent dyes, a repetitive analysis of microcirculation in the auricle can easily be performed, without the need for surgical preparation. Furthermore, this model can be adapted for in vivo studies of different issues, including wound healing, reperfusion injury, or angiogenesis. In summary, the ear of hairless mice is an ideal model for the in vivo study of skin microcirculation in physiological or pathophysiological conditions and for the evaluation of its reaction to different systemic or topical treatments.

The ear model of the hairless SKH1-Hrhr mouse enables intravital fluorescence microscopy of microcirculation and phototoxic thrombus induction without prior surgical preparation in the examined microvascular bed. Therefore, the ear of the hairless mouse is an excellent in vivo model to study the complex interactions during microvascular thrombus formation, thrombus evolution, and thrombolysis.

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

Real-Time Monitoring of Cellular Dynamics in Mouse Lung Transplantation

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in na&#239;ve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

Author Spotlight: Advancing Lung Transplant Immunology Through Intravital Imaging

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by ...

Imaging Neutrophil Migration in the Mouse Skin to Investigate Subcellular Membrane Remodeling Under Physiological Conditions

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first immune cells to reach the site of inflammation and to participate in the innate immune response during infection or tissue damage. So far, neutrophil migration has been successfully visualized using various in vitro experimental systems based on uniform stimulation, or confined migration under agarose, or micro-fluidic channels. However, these models do not recapitulate the complex microenvironment that neutrophils encounter in vivo. The development of multiphoton microscopy (MPM)-based techniques, such as intravital subcellular microscopy (ISMic), offer a unique tool to visualize and investigate neutrophil dynamics at subcellular resolutions under physiological conditions. In particular, the ear of a live anesthetized mouse provides an experimental advantage to follow neutrophil interstitial migration in real-time due to its ease of accessibility and lack of surgical exposure. ISMic provides the optical resolution, speed, and depth of acquisition necessary to track both cellular and, more importantly, subcellular processes in 3D over time (4D). Moreover, multi-modal imaging of the interstitial microenvironment (i.e., blood vessels, resident cells, extracellular matrix) can be readily accomplished using a combination of transgenic mice expressing select fluorescent markers, exogenous labeling via fluorescent probes, tissue intrinsic fluorescence, and second/third harmonic generated signals. This protocol describes 1) the preparation of neutrophils for adoptive transfer into the mouse ear, 2) different settings for optimal sub-cellular imaging, 3) strategies to minimize motion artifacts while maintaining a physiological response, 4) examples of membrane remodeling observed in neutrophils using ISMic, and 5) a workflow for the quantitative analysis of membrane remodeling in migrating neutrophils in vivo.

Neutrophil migration relies on the rapid and continuous remodeling of the plasma membrane in response to chemoattractant and its interactions with the extracellular microenvironment. Described herein is a procedure based on Intravital Subcellular Microscopy to investigate the dynamic of membrane remodeling in neutrophils injected in the ear of anesthetized mice.

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first ...

Biology

Intravital Imaging Using an Inverted Microscope to Track Skin Cell Dynamics

Streamlined Intravital Imaging Approach for Long-Term Monitoring of Epithelial Tissue Dynamics on an Inverted Confocal Microscope

Understanding normal and aberrant in vivo cell behaviors is necessary to develop clinical interventions to thwart disease initiation and progression. It is therefore critical to optimize imaging approaches that facilitate the observation of cell dynamics in situ, where tissue structure and composition remain unperturbed. The epidermis is the body&#39;s outermost barrier, as well as the source of the most prevalent human cancers, namely cutaneous skin carcinomas. The accessibility of skin tissue presents a unique opportunity to monitor epithelial and dermal cell behaviors in intact animals using noninvasive intravital microscopy. Nevertheless, this sophisticated imaging approach has primarily been achieved using upright multiphoton microscopes, which represent a significant barrier for entry for most investigators. This study presents a custom-designed, 3D-printed microscope stage insert suitable for use with inverted confocal microscopes, streamlining the long-term intravital imaging of ear skin in live transgenic mice. We believe this versatile invention, which may be customized to fit the inverted microscope brand and model of choice and adapted to image additional organ systems, will prove invaluable to the greater scientific research community by significantly enhancing the accessibility of intravital microscopy. This technological advancement is critical for bolstering our understanding of live cell dynamics in normal and disease contexts.

Author Spotlight: Developing a Tool for Using Inverted Confocal Microscopes for In Vivo Intravital Imaging 

The protocol presents a new tool to simplify intravital imaging using inverted confocal microscopy.

Understanding normal and aberrant in vivo cell behaviors is necessary to develop clinical interventions to thwart disease initiation and progression. It ...

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

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