The authors thank the members of the Whiteheart Laboratory for their careful perusal of this manuscript. The work was supported by grants from the NIH, NHLBI (HL56652, HL138179, and HL150818), and a Department of Veterans Affairs Merit Award to S.W.W., R01 HL 155519 to B.S., and NIBIB intramural program grant to R.D.L.

All experiments discussed here were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Kentucky.
NOTE: Surgical instruments are listed in Figure 1 and the Table of Materials. C57BL/6J mice, 8-10 weeks old, male/female or relevant genetically manipulated (Knockout or Knockin) strains were used.
1. FeCl3-induced carotid artery injury
<ol>
	<li>Mouse anesthesia induction
	<ol>
		<li>Weigh the mouse.</li>
		<li>Anesthetize the mouse by injecting 0.2 g/kg tribromoethanol solution intraperitoneally (i.p.). Make sure that the anesthetic solution is at room temperature (RT) before injecting it. This dose is enough to sedate the mouse for about 1 h.</li>
		<li>Check the toe reflex by pinching the toe 5 min after injection to make sure the mouse is sedated. If the mouse is not sedated, it will pull its toe away. If that happens, wait 5 min and check again.</li>
		<li>If the mouse is still not completely sedated, administer 1/4 of the initial dose and wait for 5 min.</li>
		<li>Throughout the procedure, periodically monitor the anesthetic plane of the mouse via a toe-pinch. Additionally, respiration rate and body temperature could also be monitored to maintain optimum anesthesia.</li>
	</ol>
	</li>
	<li>Immobilize the mouse
	<ol>
		<li>Lay the mouse on the heating pad (37 &#176;C) in a supine position&#160;and use adhesive tape to immobilize the extremities.</li>
		<li>Use a surgical thread (0.1 mm) to gently pull the upper front teeth of the mouse to extend the cervical/neck region. This is important for better visualization of the surgical area and stability of the Doppler probe. The thread size is not important, as it is only used to pull the front teeth of the animal to extend the neck.</li>
	</ol>
	</li>
	<li>Incision
	<ol>
		<li>Spray the surgical area with 70% ethanol or wipe it with an alcohol wipe.</li>
		<li>Using a cotton swab, apply a small amount of hair-removal cream on the surgical area. Wait for 1-2 min and then use a wet paper towel or tissue to remove the cream. 
		NOTE: This step is important for a clean surgical area.</li>
		<li>Using scissors and forceps (Figure 1B11,1B13), make a midline incision from the mandible to the suprasternal notch. Retract the skin to obtain a better visualization of the area (Figure 2A).</li>
	</ol>
	</li>
	<li>Carotid artery exposure
	<ol>
		<li>Using surgical forceps and micro dissecting forceps, blunt dissect the overlaying superficial fascia and remove it to expose the left carotid artery (Figure 1B12,1B13). Make sure not to use scissors at this stage to avoid injuring other vasculature in this area.</li>
		<li>Remove extra tissue surrounding the carotid artery. Avoid excessive dissection of the surrounding tissue, since this area harbors the vagus nerve and vertebral artery.</li>
		<li>Separate the carotid artery from surrounding tissue by dissecting it with surgical and suture-tying forceps (Figure 1B12,1B15,2C). Make sure not to extend or pull the artery to avoid any mechanical injury to the artery or the surrounding vasculature.</li>
		<li>Place a piece of plastic under the artery to mark the location of the injury (Figure 2D). Use a colored transparency sheet to make it easier to see in the surgical field.</li>
	</ol>
	</li>
	<li>Placement of the flow probe
	<ol>
		<li>Place the Doppler transonic flow probe in saline solution (0.9% NaCl in dH2O) for about 10 min before the surgery. Insert the other end of the probe into an attachment in the flowmeter to facilitate the reading of the flow. Attach the flowmeter to a computer. 
		NOTE: In between surgeries, the Doppler transonic flow probe should be in saline. Make sure to keep the probe moist and clean throughout the procedure.</li>
		<li>Place the ultrasound Doppler transonic flow probe around the artery upstream of the plastic (Figure 2D). Make sure that the vessel holder region of the probe is not too extended (Figure 1C, red arrow). 
		NOTE: If needed, support the probe with gauze pads (Figure 1B1) to achieve an appropriate height of the probe, facilitating the optimal reading position.</li>
		<li>If the surgical area is dry by this point, add a few drops of RT saline to keep the area moist. Monitor the flow probe reading. The optimal reading varies for each animal and could be anywhere between 0.6-1.2 mL/min. If it is less or not stable, change the flow probe position to get an optimal reading. 
		NOTE: Make sure that the neck of the mouse is not too extended, and that the surgical area is clean.</li>
	</ol>
	</li>
	<li>Record the flow baseline
	<ol>
		<li>After placing the probe, monitor the blood flow for 2 min to ensure that the flow is steady. Then, record the flow for 2 min (Figure 3B) as a baseline. For a detailed description of the flowmeter, refer to Subramaniam et al.17.</li>
		<li>To record the blood flow, start the WinDAQ software. Click on the File option and then click on Record. Make a new folder and file, and start recording. To stop recording, click on Stop in the file section. 
		NOTE: WinDAQ software is freely available from the publisher:&#160;https://www.dataq.com/products/windaq/.</li>
	</ol>
	</li>
	<li>Injury
	<ol>
		<li>Stop the recording of the flow. Make sure not to alter the probe position so that the readings stay consistent after the injury.</li>
		<li>Dry the area thoroughly with a wipe/paper towel. 
		NOTE: Even a small volume of residual liquid may alter the concentration of FeCl3&#160;solution used to inflict vascular injury. This leads to variable results and affects reproducibility. When the area is completely dry, the probe is not&#160;able to measure the flow.</li>
		<li>In a plastic weighing boat, put a circular filter paper (1 mm diameter, cut with a 1 mm diameter ear punch). Add 1 &#181;L of 6% FeCl3 solution (made in dH2O) onto the filter paper. 
		NOTE: Make sure to add FeCl3&#160;solution just before using the filter paper. Soaking the paper too early may lead to the evaporation of FeCl3&#160;solution and change the severity of the injury.</li>
		<li>Using fine forceps, pick up the filter paper and put it on the artery upstream of the probe, on the part of the artery marked by the plastic (Figure 2D,E). This plastic acts as a marker and a spacer. It marks the injured area and prevents accidental dilution of the FeCl3 by avoiding contact with the surrounding moist area. 
		&#8203;NOTE: Make sure that the filter paper is straight, not pinched or otherwise folded in any way. Once placed on the artery, do not change the position of the filter paper; doing so changes the extent of the injury.</li>
		<li>After placing the filter paper, start the timer and record the blood flow. After 3 min, remove the paper and add saline at the site of injury to remove the FeCl3 and stop the injury process. It also makes the area moist. At this stage, the blood flow should return to the baseline level, as recorded pre-injury.</li>
		<li>Monitor and record the flow till it reduces to 10% of the original recording or reaches zero. There is a steady decrease once the thrombus starts to form at the injury site (Figure 3C). Note any significant fluctuations in the flow during this time.</li>
		<li>To study the stability of the thrombus, record the rapid increase in blood flow after each significant and consistent decrease. These events are classified as embolization due to unstable thrombosis (Figure 4D). After the consistent decrease in flow, the operator is able to see the injury on the artery at the termination of the experiment (Figure 2F, blue arrow/dotted oval).</li>
		<li>The vessel occlusion time is defined as the complete cessation of blood flow for at least 1 min. Record the time at which an occlusive thrombus is formed, as shown by the zero reading or significant decrease in flow.</li>
		<li>Terminate the experiment at 30 min. If the thrombus does not form within 30 min of monitoring, then record the flow, stop the recording, and remove the probe.</li>
		<li>Clean the probe with 70% ethanol and a brush to remove any residual tissue/hair or debris. Dry the probe completely before placing it in the storage box.&#160;Dry the probe completely before placing it in the storage box for long term storage.</li>
		<li>Euthanize the mouse by cervical dislocation. Place the carcass in the carcass disposal bag, labeled with the lab&#39;s name and the date.</li>
	</ol>
	</li>
</ol>

2. Collection and preparation of samples for serial block face scanning electron microscopy (SBF-SEM) studies post FeCl3-induced Injury
NOTE: The EM protocol presented is appropriate for sample preparation for SBF-SEM. This imaging technique offers an unprecedented ability to study the three-dimensional structure of platelets in a clot. With this technique, the sample is visualized as a series of sequential block SEM images, generated as one progresses through the sample. Key points for this preparation are: 1) the sample must be stained with heavy metals pre-embedding; and 2) the plastic embedded sample must be trimmed appropriately for mounting within the SEM (see Figure 6 for a visual of trimmed samples). Post-embedding staining is not possible within the SEM. When collecting a sample from a FeCl3-induced vessel injury for EM analysis, the following changes need to be made while performing the surgery. The modifications in the FeCl3 surgery protocol presented are also applicable to any form of electron microscopy. Anesthesia conditions and the steps for making an incision and exposing the carotid artery are the same as in Protocol 1.
<ol>
	<li>After exposing the carotid artery, mark the site of injury by loosely encircling the region between two surgical threads (size: 0.1 mm) (Figure 5A,B).</li>
	<li>Place the probe under the artery, distal from the lower thread (Figure 5C).</li>
	<li>Insert the plastic piece under the artery between the two threads to mark the site for FeCl3 injury (Figure 5C).</li>
	<li>Take flow measurements before performing injury to note the basal flow. These measurements are used to decide at which stage the sample will be collected. 
	NOTE: Make sure the fixative (3% paraformaldehyde/PFA + 0.1% glutaraldehyde, both made in 1x PBS) is ready and at RT.&#160;Alternatively, fixation with 2.5% glutaraldehyde in a saline background could also be used. 
	CAUTION: Both paraformaldehyde and glutaraldehyde are highly toxic and irritant. While handling them, gloves, an eye shield, and masks should be used to protect from exposure. All fixative solutions are toxic, and fixation steps should be carried out in a fume hood.</li>
	<li>Perform the injury by placing FeCl3-soaked filter paper on the artery (8% FeCl3 is used) for 3 min (this time could vary as well). After 3 min, remove the filter paper and add saline at the injury site to remove any excess residual FeCl3. This step is necessary to prevent the variable extent of the injury and to facilitate flow counting by the probe (Figure 5D).</li>
	<li>Monitor the flow reading. When the flow drops below 50% of the initial value, remove the probe. Quickly dry the area and add the fixative in the area to externally fix the injury area.</li>
	<li>Promptly hold the artery near the injury area with forceps, and cut downstream of the lower thread and upstream of the upper thread. If necessary, ask another person to help cut one end. Put the tissue on the plastic tissue culture dish in the same orientation as it was collected, and add a few drops of the fixative. 
	NOTE: Cutting the artery causes immediate extensive bleeding, so make sure to hold the artery before cutting it the first time. In this scenario, the mouse is exsanguinated. The mouse is euthanized by exsanguination and cervical dislocation.</li>
	<li>Using a scalpel, clean the extra tissue around the artery. Be mindful to record the orientation of the blood flow. To mark that, cut one end horizontally and the other tapering/oblique (Figure 5E).</li>
	<li>Keep the sample in fixative (3% PFA and 0.1% glutaraldehyde) for 1 h at RT. Then, store the sample in 1% PFA at 4 &#176;C until sending it on wet ice to the sample processing lab for EM analysis. 
	NOTE: The samples should not be frozen. The challenges for this method of sample collection are:
	<ol>
		<li>The initial reading may not be optimal or may fluctuate after the injury. This may affect the extent of injury that is chosen to arrest for EM analysis. To address this problem, make sure to record the baseline reading for a few minutes, after trying various positions of placing the probe to decide which position is optimal.</li>
		<li>The time to arrest the injury by adding external fixative and actual cutting of the arterial section for analysis may add variability to the extent of thrombosis. Be quick during sample collection after fixing externally.</li>
	</ol>
	</li>
	<li>Receive the samples for processing for EM analysis in 1% PFA on wet ice. Rinse the samples for 3 min, on ice, with 0.1 M cacodylate buffer to initiate further processing.</li>
	<li>Fix the samples for 1 h on ice in 0.1 M cacodylate buffer + 2.5% glutaraldehyde + 2 mM CaCl2.</li>
	<li>Discard the fixative and wash the samples with cold 0.1 M sodium cacodylate buffer containing 2 mM CaCl2 (5 x 3 min) (Figure 6A).</li>
	<li>Fix the samples with osmium solution (3% potassium ferrocyanide [K4C6N6Fe] + 0.3 M cacodylate buffer + 4 mM CaCl2 mixed with an equal volume of 4% aqueous osmium tetroxide [OsO4; in ddH2O]) for 1 h on ice.</li>
	<li>While the samples are fixing, make fresh 1% trichloroacetaldehyde hydrate (TCH) solution in ddH2O. Incubate the solution at 60 &#176;C&#160;for 1 h while gently mixing.</li>
	<li>After fixing, wash the samples with ddH2O at RT for 5 x 3 min.</li>
	<li>Incubate the samples in filtered 1% TCH solution (made in ddH2O) for 20 min at RT.</li>
	<li>Wash the samples with ddH2O at RT (5 x 3 min).</li>
	<li>Fix the samples in 2% osmium tetroxide in ddH2O for 30 min at RT.</li>
	<li>Wash the samples with ddH2O at RT for 5 x 3 min each.</li>
	<li>Place the samples in 1% uranyl acetate (aqueous) and incubate overnight at 4 &#176;C.</li>
	<li>Wash the samples with ddH2O at RT for 5 x 3 min each, and process them with en bloc Walton&#39;s lead aspartate staining.</li>
	<li>En bloc Walton&#39;s lead aspartate staining3:
	<ol>
		<li>Make 30 mM L-aspartic acid solution in ddH2O. 
		NOTE: The aspartic acid dissolves more quickly if the pH is raised to 3.8. This stock solution is stable for 1-2 months if refrigerated.</li>
		<li>Make 20 mM lead nitrate solution in 10 mL of aspartic acid stock, and adjust the pH to 5.5 with 1 N KOH.</li>
		<li>Place the samples in lead aspartate solution at 60 &#176;C for 30 min, following five washes with ddH2O at RT, each for 3 min.</li>
	</ol>
	</li>
	<li>Dehydrate the specimens with a graded ethanol series. Use the chemical dehydration by Valdivia protocol18.
	<ol>
		<li>Wash the samples in each of the following solutions to progressively dehydrate the sample (Figure 6B): 
		25% ethyl alcohol for 3 min 
		50% ethyl alcohol for 3 min 
		75% ethyl alcohol for 3 min 
		95% ethyl alcohol for 3 min 
		100%&#160;ethyl alcohol for 10 min, and repeat the step twice (total of three washes).</li>
	</ol>
	</li>
	<li>Wash the samples in 100% propylene oxide (PO) for 10 min, and repeat the step twice (total of three washes).</li>
	<li>Incubate in 50%/50% PO/resin with DMP 30 activator overnight at RT, and embed in 100% Araldite 502/embed 812/DDSA with DMP30 activator for 48 h.</li>
</ol>

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<li>Woollard, K. J., Sturgeon, S., Chin-Dusting, J. P. F., Salem, H. H., Jackson, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Erythrocyte+hemolysis+and+hemoglobin+oxidation+promote+ferric+chloride-induced+vascular+injury%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Erythrocyte hemolysis and hemoglobin oxidation promote ferric chloride-induced vascular injury.</a> Journal of Biological Chemistry. 284 (19), 13110-13118 (2009).</li>
<li>Shim, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+ferric+chloride-induced+arterial+thrombosis+model+of+mice+and+the+role+of+red+blood+cells+in+thrombosis+acceleration%5BTitle%5D)+AND+%22Yonsei+Medical+Journal%22%5BJournal%5D)">Characterization of ferric chloride-induced arterial thrombosis model of mice and the role of red blood cells in thrombosis acceleration.</a> Yonsei Medical Journal. 62 (11), 1032-1041 (2021).</li>
<li>Ghosh, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+the+prothrombotic+potential+of+four-factor+prothrombin+complex+concentrate+%284F-PCC%29+in+animal+models%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Evaluation of the prothrombotic potential of four-factor prothrombin complex concentrate (4F-PCC) in animal models.</a> PLoS One. 16 (10), 0258192(2021).</li>
<li>Wilbs, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cyclic+peptide+FXII+inhibitor+provides+safe+anticoagulation+in+a+thrombosis+model+and+in+artificial+lungs%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Cyclic peptide FXII inhibitor provides safe anticoagulation in a thrombosis model and in artificial lungs.</a> Nature Communications. 11 (1), 3890(2020).</li>
<li>Wei, Y., Deng, X., Sheng, G., Guo, X. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+rabbit+model+of+cerebral+venous+sinus+thrombosis+established+by+ferric+chloride+and+thrombin+injection%5BTitle%5D)+AND+%22Neuroscience+Letters%22%5BJournal%5D)">A rabbit model of cerebral venous sinus thrombosis established by ferric chloride and thrombin injection.</a> Neuroscience Letters. 662, 205-212 (2018).</li>
<li>Jacob-Ferreira, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antithrombotic+activity+of+Batroxase%2C+a+metalloprotease+from+Bothrops+atrox+venom%2C+in+a+model+of+venous+thrombosis%5BTitle%5D)+AND+%22International+Journal+of+Biological+Macromolecules%22%5BJournal%5D)">Antithrombotic activity of Batroxase, a metalloprotease from Bothrops atrox venom, in a model of venous thrombosis.</a> International Journal of Biological Macromolecules. 95, 263-267 (2017).</li>
<li>Zhou, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+rabbit+model+of+cerebral+microembolic+signals+for+translational+research%3A+preclinical+validation+for+aspirin+and+clopidogrel%5BTitle%5D)+AND+%22Journal+of+Thrombosis+and+Haemostasis%22%5BJournal%5D)">A rabbit model of cerebral microembolic signals for translational research: preclinical validation for aspirin and clopidogrel.</a> Journal of Thrombosis and Haemostasis. 14 (9), 1855-1866 (2016).</li>
<li>Yang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+evodiamine+on+collagen-induced+platelet+activation+and+thrombosis%5BTitle%5D)+AND+%22BioMed+Research+International%22%5BJournal%5D)">Effect of evodiamine on collagen-induced platelet activation and thrombosis.</a> BioMed Research International. 2022, 4893859(2022).</li>
<li>Li, W., McIntyre, T. M., Silverstein, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ferric+chloride-induced+murine+carotid+arterial+injury%3A+A+model+of+redox+pathology%5BTitle%5D)+AND+%22Redox+Biology%22%5BJournal%5D)">Ferric chloride-induced murine carotid arterial injury: A model of redox pathology.</a> Redox Biology. 1 (1), 50-55 (2013).</li>
<li>Li, W., Nieman, M., Sen Gupta, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ferric+chloride-induced+murine+thrombosis+models%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Ferric chloride-induced murine thrombosis models.</a> Journal of Visualized Experiments. (115), e54479(2016).</li>
<li>Holly, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ether+lipid+metabolism+by+AADACL1+regulates+platelet+function+and+thrombosis%5BTitle%5D)+AND+%22Blood+Advances%22%5BJournal%5D)">Ether lipid metabolism by AADACL1 regulates platelet function and thrombosis.</a> Blood Advances. 3 (22), 3818-3828 (2019).</li>
<li>Bird, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prediction+of+the+therapeutic+index+of+marketed+anti-coagulants+and+anti-platelet+agents+by+guinea+pig+models+of+thrombosis+and+hemostasis%5BTitle%5D)+AND+%22Thrombosis+Research%22%5BJournal%5D)">Prediction of the therapeutic index of marketed anti-coagulants and anti-platelet agents by guinea pig models of thrombosis and hemostasis.</a> Thrombosis Research. 123 (1), 146-158 (2008).</li>
</ol>

The authors have no conflicts of interest related to this study.
ORCID profiles: S.J.: 0000-0001-6925-2116; S.W.W.: 00000-0001-5577-0473.

The topical application of FeCl3 to the vasculature to induce thrombosis is a widely used technique, and has been instrumental in establishing roles for various platelet receptors, ligand signaling pathways, and their inhibitors20,21,22,23. The mechanism through which FeCl3 causes thrombosis is multifaceted; previously, endothelial denudation was considered a cause of thro...

Thrombosis is the formation of a blood clot that partially or completely blocks a blood vessel, impeding the natural flow of the blood. This leads to severe and fatal cardiovascular events, such as ischemic heart disease and strokes. Cardiovascular diseases are the leading cause of morbidity and mortality, and cause one in four deaths worldwide1,2,3. Although thrombosis is manifested as a malfunction of the vascular system, it could be a result of an underlying microbial or viral infection, immune disorder, malignancy, or metabolic condition. The flow of blood is maintained by the complex interaction among diverse components of the vascular system, including endothelial cells, red/white blood cells, platelets, and coagulation factors4. Upon vascular injury, platelets interact with adhesive proteins on the subendothelial matrix and release their granular contents, which recruit more platelets5. Concurrently, the coagulation cascade is activated, leading to fibrin formation and deposition. Ultimately, a clot is formed, containing platelets and red blood cells trapped within a fibrin mesh6. Although antiplatelet and anticoagulant drugs are available to modulate thrombosis, spurious bleeding remains a major concern with these therapies, requiring fine-tuning of the dosages and combinations of these drugs. Thus, there is still an urgent need to discover new anti-thrombotic drugs7.
Thrombosis is studied using multiple methods to inflict vascular injury: mechanical (vessel ligation), thermal (laser injury), and chemical injury (FeCl3/Rose Bengal application). The nature of thrombosis varies depending on the location (arterial vs. venous), method, or extent of the injury. Among all these types, FeCl3-induced vascular injury is the most widely used method. It has been employed in mice, rats, rabbits, guinea pigs, and dogs8,9,10,11,12. The method is relatively simple, easy to use, and if major parameters are standardized, it is sensitive and reproducible in various vascular systems (e.g., arteries [carotid and femoral], veins [jugular], and arterioles [cremaster and mesenteric]) (Supplemental Table 1).
This model can also be used to further our understanding of the mechanics and morphology of clot formation. This technique uniquely offers the advantage of stopping thrombosis at various flow rate points, to study the intermediate stages of the process before it becomes occlusive. Recent advances in thrombosis research have used this model to focus attention on non-pharmacological methods of thrombolysis13 or non-invasive delivery of anti-thrombotic and/or fibrinolytic agents14,15. Several groups have shown that, when platelet membranes are coated with these therapeutics, the drugs can be activated upon thermal stimulation to target clots16. The techniques described here can be useful to such studies as validation of their findings at the single platelet level. In this manuscript, Protocol 1 describes the basic FeCl3-mediated vascular injury procedure, while Protocol 2 describes the method to collect and fix the vascular injury sample for further analysis by electron microscopy.

<table><tbody><tr><td>0.9% Saline&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>BP358-212</td><td>NaCl used to make a solution of 0.9% saline&amp;nbsp;</td></tr><tr><td>1 mL Syringe&amp;nbsp;</td><td>Becton, Dickinson and Company&amp;nbsp;</td><td>309659</td><td></td></tr><tr><td>190 Proof Ethanol&amp;nbsp;</td><td>KOPTEC</td><td>V1101&amp;nbsp;</td><td>Used to make a 70% ethanol solution to use for prepping the mouse for surgery&amp;nbsp;</td></tr><tr><td>2,2,2 Tribromoethanol</td><td>Sigma Aldrich</td><td>48402</td><td></td></tr><tr><td>25 Yard Black Braided Silk Suture (5-0)</td><td>DEKNATEL</td><td>136082-1204</td><td></td></tr><tr><td>26G x 3/8 Needle&amp;nbsp;</td><td>Becton, Dickinson and Company&amp;nbsp;</td><td>305110</td><td></td></tr><tr><td>2-methyl-2-butanol</td><td>Sigma Aldrich</td><td>240486</td><td></td></tr><tr><td>7.5 mL Transfer Pipet, Graduated to 3 mL</td><td>Globe Scientific Inc.</td><td>135010</td><td></td></tr><tr><td>Alcohol Prep Pads (70% Isopropyl Alcohol)</td><td>Medline</td><td>MDS090735</td><td></td></tr><tr><td>Araldite GY 502&amp;nbsp;</td><td>Electron microscopy Services&amp;nbsp;</td><td>10900</td><td></td></tr><tr><td>Cell Culture Dish 35mm X 10mm&amp;nbsp;</td><td>Corning Incorporated&amp;nbsp;</td><td>430165</td><td></td></tr><tr><td>Compact Scale&amp;nbsp;</td><td>Ward&#039;s Science&amp;nbsp;</td><td>470314-390</td><td></td></tr><tr><td>Dissecting Scissors, 12.5 cm long</td><td>World Precision Instrument</td><td>15922-G</td><td></td></tr><tr><td>DMP-30 activator&amp;nbsp;</td><td>Electron microscopy Services&amp;nbsp;</td><td>13600</td><td></td></tr><tr><td>Dodenyl Succinic Anhydride/ DDSA</td><td>Electron microscopy Services&amp;nbsp;</td><td>13700</td><td></td></tr><tr><td>Doggy Poo Bags/animal carcass disposal bag</td><td>Crown Products&amp;nbsp;</td><td>PP-RB-200</td><td></td></tr><tr><td>Doppler FlowProbe</td><td>Transonic Systems Inc.</td><td>MA0.5PSB</td><td></td></tr><tr><td>EMBED 812 resin&amp;nbsp;</td><td>Electron microscopy Services&amp;nbsp;</td><td>14900</td><td></td></tr><tr><td>Ethyl Alcohol, anhydrous 200 proof&amp;nbsp;</td><td>Electron microscopy Services&amp;nbsp;</td><td>15055</td><td></td></tr><tr><td>Eye Dressing Forceps, 4&quot; 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1x2 Teeth, Full Curve; 0.8 mm Tip Width; 4&quot; Length</td><td>Roboz Surgical Instrument Company</td><td>RS-5157</td><td></td></tr><tr><td>Osmium Tetroxide 4% aqueous solution&amp;nbsp;</td><td>Electron microscopy Services&amp;nbsp;</td><td>19150</td><td></td></tr><tr><td>Paraformaldehyde (16% solution)</td><td>Electron microscopy Services&amp;nbsp;</td><td>15710</td><td></td></tr><tr><td>Potassium ferricyanide</td><td>SIGMA-ALDRICH</td><td>P-8131</td><td></td></tr><tr><td>Propylene Oxide, ACS reagent&amp;nbsp;</td><td>Electron microscopy Services&amp;nbsp;</td><td>20401</td><td></td></tr><tr><td>Rainin Classic Pipette PR-10</td><td>Rainin</td><td>17008649</td><td></td></tr><tr><td>Research Flowmeter&amp;nbsp;</td><td>Transonic Systems Inc.</td><td>T402B01481</td><td>Model: T402</td></tr><tr><td>Scotch Magic Invisible Tape, 3/4&quot; x 1000&quot;, Clear</td><td>Scotch&amp;nbsp;</td><td>305289</td><td></td></tr><tr><td>Small Animal Heated Pad</td><td>K&amp;amp;H Manufacturing Inc.</td><td>Model: HM10</td><td></td></tr><tr><td>Sodium Cacodylate Buffer 0.2M, pH7.4</td><td>Electron microscopy Services&amp;nbsp;</td><td>11623</td><td></td></tr><tr><td>Sterile Cotton Tipped Applicators&amp;nbsp;</td><td>Puritan Medical Products&amp;nbsp;</td><td>25-806 1WC</td><td></td></tr><tr><td>Steromaster Illuminator&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>12-562-21</td><td>No longer available from the company</td></tr><tr><td>Surgical Dumont #7 Forceps&amp;nbsp;</td><td>Fine Science Tools&amp;nbsp;</td><td>11271-30</td><td></td></tr><tr><td>Thiocarbohydrazide (TCH)</td><td>SIGMA-ALDRICH</td><td>88535</td><td></td></tr><tr><td>Universal Low Retention Pipet Tip Reloads (0.1-10 &amp;micro;L)</td><td>VWR</td><td>76323-394</td><td></td></tr><tr><td>Uranyl Acetate</td><td>Electron microscopy Services&amp;nbsp;</td><td>22400</td><td></td></tr><tr><td>Veet Gel Cream Hair Remover</td><td>Reckitt Benckiser</td><td>3116875</td><td></td></tr><tr><td>White Antistatic Hexagonal Weigh Boats, Medium, 64 x 15 x 19 mm</td><td>Fisher Scientific&amp;nbsp;</td><td>S38975</td><td></td></tr><tr><td>WinDAQ/100 Software for Windows</td><td>DATAQ Instruments, Inc.</td><td>Version 3.38</td><td>Freely available to download. https://www.dataq.com/products/windaq/</td></tr><tr><td>ZEISS AxioCam Icc 1</td><td>ZEISS</td><td>57615</td><td></td></tr></tbody></table>

ferric chloride-induced arterial thrombosis and sample collection for 3d electron microscopy analysis

Cardiovascular diseases are a leading cause of mortality and morbidity worldwide. Aberrant thrombosis is a common feature of systemic conditions like diabetes and obesity, and chronic inflammatory diseases like atherosclerosis, cancer, and autoimmune diseases. Upon vascular injury, usually the coagulation system, platelets, and endothelium act in an orchestrated manner to prevent bleeding by forming a clot at the site of the injury. Abnormalities in this process lead to either excessive bleeding or uncontrolled thrombosis/insufficient antithrombotic activity, which translates into vessel occlusion and its sequelae. The FeCl3-induced carotid injury model is a valuable tool in probing how thrombosis initiates and progresses in vivo. This model involves endothelial damage/denudation and subsequent clot formation at the injured site. It provides a highly sensitive, quantitative assay to monitor vascular damage and clot formation in response to different degrees of vascular damage. Once optimized, this standard technique can be used to study the molecular mechanisms underlying thrombosis, as well as the ultrastructural changes in platelets in a growing thrombus. This assay is also useful to study the efficacy of antithrombotic and antiplatelet agents. This article explains how to initiate and monitor FeCl3-induced arterial thrombosis and how to collect samples for analysis by electron microscopy.

The data are generally presented as time to occlusion, or time required to form a fully occlusive thrombus. These data can be plotted as a Kaplan-Meier survival curve (Figure 4A)19, a dot plot with bars showing the terminal blood flow at the time of either cessation of the blood flow or the termination of an experiment (Figure 4B), or as a line graph (Figure 4C). Thrombus stability can be studied using this t...

Watch this Scientific Journal Video about Ferric Chloride-Induced Arterial Thrombosis and Sample Collection for 3D Electron Microscopy Analysis at JoVE.com

Ferric Chloride-Induced Arterial Thrombosis and Sample Collection for 3D Electron Microscopy Analysis

The present protocol describes how to use a FeCl3-mediated injury to induce arterial thrombosis, and how to collect and prepare arterial injury samples at various stages of thrombosis for electron microscopy analysis.

Cardiovascular diseases are a leading cause of mortality and morbidity worldwide. Aberrant thrombosis is a common feature of systemic conditions like ...

ferric-chloride-induced-arterial-thrombosis-sample-collection-for-3d

Research

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Medicine

1.8K Views.  University of Kentucky. The present protocol describes how to use a FeCl3-mediated injury to induce arterial thrombosis, and how to collect and prepare arterial injury samples at various stages of thrombosis for electron microscopy analysis.

Video: Ferric Chloride-Induced Arterial Thrombosis and Sample Collection for 3D Electron Microscopy Analysis

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A&#946;-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A&#946; and A&#946;-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A&#946;-fibrinogen interaction inhibitor.

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

Biochemistry

Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. The feasibility of using magnetic forces to capture and retain endothelial outgrowth cells (EOC) labeled with super paramagnetic iron oxide nanoparticles (SPION) has been shown previously. But this technique requires the development of a mechanically functional stent from a magnetic and biocompatible material followed by in-vitro and in-vivo testing to prove rapid endothelialization. We developed a weakly ferromagnetic stent from 2205 duplex stainless steel using computer aided design (CAD) and its design was further refined using finite element analysis (FEA). The final design of the stent exhibited a principal strain below the fracture limit of the material during mechanical crimping and expansion. One hundred stents were manufactured and a subset of them was used for mechanical testing, retained magnetic field measurements, in-vitro cell capture studies, and in-vivo implantation studies. Ten stents were tested for deployment to verify if they sustained crimping and expansion cycle without failure. Another 10 stents were magnetized using a strong neodymium magnet and their retained magnetic field was measured. The stents showed that the retained magnetism was sufficient to capture SPION-labeled EOC in our in-vitro studies. SPION-labeled EOC capture and retention was verified in large animal models by implanting 1 magnetized stent and 1 non-magnetized control stent in each of 4 pigs. The stented arteries were explanted after 7 days and analyzed histologically. The weakly magnetic stents developed in this study were capable of attracting and retaining SPION-labeled endothelial cells which can promote rapid healing.

Our goals were to design, manufacture and test ferromagnetic stents for endothelial cell capture. Ten stents were tested for fracture and 10 more stents were tested for retained magnetism. Finally, 10 stents were tested in-vitro and 8 more stents were implanted in 4 pigs to show cell capture and retention.

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. ...

Bioengineering

A Fibrinolytic Enzyme-Based Method to Study Cerebral Thrombus Cell Components

A Comprehensive Approach to Analyze the Cell Components of Cerebral Blood Clots

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of cerebral blood clots is important for diagnosis, treatment, and prognosis. However, the current approaches to studying the cell components of the clots are mainly based on in situ staining, which is unsuitable for the comprehensive study of the cell components because cells are tightly wrapped in the clots. Previous studies have successfully isolated a fibrinolytic enzyme (sFE) from Sipunculus nudus, which can degrade the cross-linked fibrin directly, releasing the cell components. This study established a comprehensive method based on the sFE to study the cell components of cerebral thrombus. This protocol includes clot dissolving, cell releasing, cell staining, and routine blood examination. According to this method, the cell components could be studied quantitatively and qualitatively. The representative results of experiments using this method are shown.

Author Spotlight: A Novel Method for Comprehensive Cell Component Analysis of Cerebral Blood Clots 

This study describes a fast and effective method for the cell component analysis of cerebral blood clots through clot dissolving, cell staining, and routine blood examination.

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of ...

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Ferric Chloride-induced Murine Thrombosis Models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...

Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, we have translated a vascular injury model from a small animal into a large animal (canine), with slight modifications that can be used for pre-clinical screening of prophylactic and thrombolytic agents. In addition to the surgical methods, the modified protocol describes the step-by-step methods to assess carotid artery canalization by angiography, detailed instructions to process both the brain and carotid artery for histological analysis to verify carotid canalization and cerebral hemorrhage, and specific parameters to complete an assessment of downstream thromboembolic events by utilizing magnetic resonance imaging (MRI). In addition, specific procedural changes from the previously well-established small animal model necessary to translate into a large animal (canine) vascular injury are discussed.

Here, we present the modifications necessary to a well characterized and commonly used small animal ferric chloride-induced (FeCl3) carotid artery injury model for use in a large animal vascular injury model. The resulting model can be utilized for pre-clinical trial assessment of both prophylactic and thrombolytic pharmacological and mechanical interventions.

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, ...

Puncture Wound Hemostasis and Montaged Electron Microscopy 

Puncture Wound Hemostasis and Preparation of Samples for Montaged Wide-Area Electron Microscopy Analysis

Hemostasis, the process of normal physiological control of vascular damage, is fundamental to human life. We all suffer minor cuts and puncture wounds from time to time. In hemostasis, self-limiting platelet aggregation leads to the formation of a structured thrombus in which bleeding cessation comes from capping the hole from the outside. Detailed characterization of this structure could lead to distinctions between hemostasis and thrombosis, a case of excessive platelet aggregation leading to occlusive clotting. An imaging-based approach to puncture wound thrombus structure is presented here that draws upon the ability of thin-section electron microscopy to visualize the interior of hemostatic thrombi. The most basic step in any imaging-based experimental protocol is good sample preparation. The protocol provides detailed procedures for preparing puncture wounds and platelet-rich thrombi in mice for subsequent electron microscopy. A detailed procedure is given for in situ fixation of the forming puncture wound thrombus and its subsequent processing for staining and embedding for electron microscopy. Electron microscopy is presented as the end imaging technique because of its ability, when combined with sequential sectioning, to visualize the details of the thrombus interior at high resolution. As an imaging method, electron microscopy gives unbiased sampling and an experimental output that scales from nanometer to millimeters in 2 or 3 dimensions. Appropriate freeware electron microscopy software is cited that will support wide-area electron microscopy in which hundreds of frames can be blended to give nanometer-scale imaging of entire puncture wound thrombi cross-sections. Hence, any subregion of the image file can be placed easily into the context of the full cross-section.

Author Spotlight: Revealing Platelet Dynamics Through Advances in Structural Hematology

A puncture wound procedure for hemostatic thrombus formation is presented here. The formed thrombi are large and are hundreds of microns in diameter. Hence, volume imaging approaches are appropriate. We suggest montaged wide-area transmission electron microscopy as a high-resolution approach available to many and detail a preparative protocol.

Hemostasis, the process of normal physiological control of vascular damage, is fundamental to human life. We all suffer minor cuts and puncture wounds ...

Intravital Fluorescence Microscopy to Study Microvascular Thrombus Formation

Source: Str&#252;der, D., et al. <a href="https://app.jove.com/v/55174">Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse.</a> J. Vis. Exp. (2017)
This video demonstrates the intravital fluorescence microscopy technique to study thrombus formation in a mouse model. The mouse is injected with a fluorophore-labeled polysaccharide to monitor blood circulation. Upon illumination with a high-intensity light, the phototoxic induction of an injury in the microvasculature of the ear leads to the formation of a thrombus, which is confirmed by visualizing the obstruction in the blood flow.