This work was supported by the Board of Regents of the State of Louisiana LEQSF(2009-12)-RD-A-19 (PI: W.L. Murfee) and the Tulane Hypertension and Renal Center of Excellence funded by NIH grant P20RR017659-08 (PI: L. Gabriel Navar).

1. Surgical Procedure Set-Up Notes
<ol>
	<li>Prior to the surgery sterilize surgical supplies and instruments. Supplies used for the sterile surgical procedure for each rat include 1 drape to be layed out as a sterile surface for the placement of instruments, one drape with a pre-cut hole about 0.5 in x 1.5 in in the center to be placed over the rat, and gauze. The pre-cut hole on the drape will align with the incision made on the rat. Instruments needed for the surgery include 1 standard pair of scissors to be used to cut suture material, two forceps and a fine needle holder for handling and gripping of the suture, and a scalpel with a blade. We have listed the common tools used by our laboratory in the Table of Specific Surgical Materials and Tools. However, final tool selection depends on experimenter preferences.</li>
	<li>Prepare the surgery space. Place a 100 mL 0.9% sterile saline bag under a heating pad to ensure that the saline that comes in contact with the mesentery tissue is pre-warmed to approximately 37 &deg;C. As an alternative to saline, you can use Ringer's solution or another physiological buffer. Lay out sterilized surgical instruments and supplies on a sterile drape for easy access during the exteriorization procedure. Also, you will need sterile cotton tip applicators and the appropriate 3 types of sutures (4-0, 5-0, and 7-0). Makes sure packages are pre-opened so that the materials can be accessed with sterile handling. Finally, disinfect the pre-modified plastic stage by immersing it in 100% ethanol for at least 5 minutes. The stage is a 100 mm Petri dish with an elliptical hole cut in the center (Figure 1). A Dremel Tool can be used to initially make and, if necessary, widen the hole. After the hole is made, the edges of the cut plastic are made smooth using sand paper of different grains. The stage is then washed and, finally, either inert modeling clay (purchased from a local crafts store) or silicon glue is added to the hole's edge in order to provide a raised, smooth surface. Prior to contact with the tissue, the stage will need to be adequately rinsed with sterile saline.</li>
	<li>For this model of angiogenesis, we typically use adult Male Wistar rats (350 &plusmn; 25 g). Other rat strains and ages can be used. In our laboratory, rats are anesthetized via intramuscular injection with ketamine (80 mg/kg b.w.), xylazine (8 mg/kg b.w.), and atropine (0.08 mg/kg b.w.). After approximately 5 mins, the effect of anesthesia is confirmed and the rat's abdominal skin is shaved.</li>
</ol>
2. Rat Mesentery Exteriorization Model
<ol>
	<li>Position the anesthetized rat on its back on a heating pad to maintain body temperature. Aseptic techniques are used during the surgical procedure. Wear sterile gloves and do not allow instruments and supplies to contact non-sterile surfaces other than the rat tissues.</li>
	<li>Clean abdominal area with alternating wipes using the sterile gauze soaked in 70% isopropyl and iodine.</li>
	<li>Using the scalpel blade, make a small (approximately 0.75 inch) longitudinal incision along the skin and then the linea alba approximately 1 inch below the sternum.</li>
	<li>Place the pre-cut drape over the abdominal section so that the opening aligns with the incision.</li>
	<li>Gently apply pressure around the incision. This usually will result in exposure of the small intestine enough for it to be easily identified.</li>
	<li>Using the cotton tip applicators, gently pull out a section of the small intestine and locate the ileum. As the mesentery is pulled out, it should be gently layed out in the plastic stage (Figure 2).</li>
	<li>Identify 6-8 vascularized mesenteric windows. A mesenteric window is defined as the thin translucent membrane in between the artery/vein pairs feeding the small intestine (Figure 2).</li>
	<li>Record the starting time of exteriorization. Leave the mesentery section exteriorized for 20 minutes. During the exteriorization period, use a sterile syringe (5 mL) to intermittently replenish the saline in the Petri dish to ensure that the tissue remains immersed and does not dry out.</li>
	<li>During the 20-minute exposure time, mark the 2 centrally located mesenteric windows with sterile 7-0 suture. The marks should be made in the fat area near the intestine.</li>
	<li>Return the exteriorized region of the mesentery to the abdominal cavity.</li>
	<li>Close the abdominal muscle with 5-0 monofilament suture in interrupted pattern.</li>
	<li>Close the skin with 4-0 monofilament suture in interrupted pattern.</li>
	<li>Wipe the sutured area with alternating wipes using the sterile gauze soaked in 70% isopropoyl and iodine.</li>
	<li>Spread eye lubricant gel on both eyes of the rat. The purpose of lubricating the eye is to prevent desiccation of the cornea during surgical procedure and recovery. The application of eye lubricant should be applied prior to the start of surgical intervention. If necessary, re-apply eye lubricant prior to returning the rat to its cage for recovery.</li>
</ol>
3. Tissue Harvesting and Fixation
<ol>
	<li>On the day of interest post stimulation, anesthetize and euthanize the rat. In our laboratory, rats are anesthetized via intramuscular injection with ketamine (80 mg/kg b.w.), xylazine (8 mg/kg b.w.), and atropine (0.8 mg/kg b.w.) and then euthanized via intracardiac injection of beuthanasia. Beuthanasia is a pentobarbital sodium/phenytoin solution that can be used for rapid and painless euthanasia. Per rat our laboratory typically injects 0.1 - 0.2 ml.</li>
	<li>Re-open the abdominal cavity and gently remove the small intestine and locate the two-marked windows.</li>
	<li>Cut out each of the exteriorized mesenteric windows using forceps and micro-scissors. Including a border of fat per window is advantageous for later tissue spreading onto microscope slides. Immediately, immerse each window in PBS. When cutting, try to avoid cutting through artery/vein pairs within the fat border of the windows to minimize potential blood filling of the windows. Also, minimize cutting through the bowel that results in intestinal content contacting the windows.</li>
	<li>Use forceps to spread tissues on positively charged microscope slides. Two tissues can be mounted per slide.</li>
	<li>Allow the tissues to partially dry and using a scalpel blade remove the excess fat.</li>
	<li>Tissues are now ready to be fixed. Typical fixation methods can be used. In our laboratory, we commonly fix the slides in methanol (-20 &deg;C) for 30 min. Successful labeling has also been performed with unfixed tissues. Fixation methods might depend on your preferred antibody.</li>
</ol>
4. Tissue Immunolabeling
<ol>
	<li>Tissues can generally now be labeled according to instructions per antibody for immunohistochemistry. Given our interest in identifying the roles of vascular pericytes during angiogenesis, our laboratory commonly labels for endothelial and perivascular cells10, 12, 13. Useful labels to identify endothelial cells along the hierarchy of the microvascular networks are an anti-PECAM antibody or BSI-lectin. Perivascular cell markers used in this tissue include NG2, desmin, SM-&alpha; actin, PDFGR&beta;, and class III &beta;-tubulin. Below, in section 3.3, we have listed protocols for colorimetric and fluorescent PECAM immunolabeling protocols.</li>
	<li>Prior to the initial primary antibody incubation, slides are vacuum dried to remove excess buffer solution. Also, tissues are initially outlined with a wax pen to prevent uncontrolled flow of antibody solutions away from the tissue. Finally, all labeling steps are followed by washing steps. For washing steps, slides are placed inside staining jars. The buffer solution is exchanged 3 times during the wash duration.</li>
	<li>Endothelial cell antibody labeling procedures:
		 PECAM Colorimetric Lableing
		<ol>
			<li>Primary antibody incubation: Drip approximately 100-200 mL primary antibody solution (1:200 mouse monoclonal biotinylated CD31 antibody diluted in antibody buffer (0.1% Saponin in PBS + 2% BSA)) on top to each tissue; make sure the whole tissue is covered with antibody solution. Incubation for 1 hour at room temperature.</li>
			<li>Wash tissues with PBS +0.1% saponin for 30 minutes.</li>
			<li>Secondary antibody incubation: Drip secondary antibody solution (VECTASTAIN Elite ABC solution from Vector Laboratories; a streptavidin peroxidase secondary antibody solution) on top of tissues. Incubate at room temperature for 1 hour.</li>
			<li>Wash tissues with PBS +0.1% saponin for 30 minutes.</li>
			<li>Incubate tissues with Vector Nova Red for 15 minutes. Then rise tissues with water.</li>
			<li>Mount slides: Dip slides in 95% ethanol 10 times. Immerse slides in 100% ethanol for 2 mins. Immerse slides in another 100% ethanol solution for 2 mins. Then immerse slides in consecutive 100% xylene solutions for 2 minutes each. Allow to dry and cover tissues with a thin layer of VectaMount and a coverslip.</li>
		</ol>
		 PECAM Fluorescent Labeling
		<ol>
			<li>Primary antibody incubation: Drip approximately 100-200 mL primary antibody solution (1:200 mouse monoclonal biotinylated CD31 antibody diluted in antibody buffer (0.1% Saponin in PBS + 2% BSA)) on top to each tissue; make sure the whole tissue is covered with antibody solution. Incubation for 1 hour at room temperature.</li>
			<li>Wash tissues with PBS +0.1% saponin for 30 minutes.</li>
			<li>Secondary antibody incubation: Drip secondary antibody solution ((1:100 Streptavidin-CY3 diluted in antibody buffer (0.1% Saponin in PBS + 2% BSA)) on top of tissues. Incubate at room temperature for 1 hour.</li>
			<li>Wash tissues with PBS +0.1% saponin for 30 minutes.</li>
			<li>Mount slides: Drip 50:50 PBS with glycerol on top of tissues. Cover with cover slip. Then, use nail polish to seal the gap between cover slip and slide.</li>
		</ol>
	</li>
</ol>
5. Representative Results
Representative images of rat mesentery tissues immunohistochemically labeled for PECAM are displayed in Figure 3. PECAM labeling identifies all vessel types along the hierarchy of remodeling microvascular networks and can be used to quantify angiogenic metrics at specific time points post stimulation. PECAM labeling also allows for the determination of arterioles versus venules. Feeding arterioles typically exhibit smaller diameters and elongated endothelial cell morphology compared to paired venules (Figure 4). Capillaries and capillary sprouts can be identified based on their vessel diameter and relative position within a network. Typical characteristics of remodeling networks include increased capillary sprouting, vessel density, vascularized area and venular tortuosity. Quantification of various angiogenic metrics identifies the time course of network growth (Figure 5). Capillary sprouting from pre-existing vessels, peaks between Day 3 and Day 5 and returns to the unstimulated level by Day 10. This transient increase in sprouting is followed by increases in vascular density and vascularized area. As evidence for the remodeling of larger vessels in this model, the number of arteriole and venule segments also increases over the time course.
In our laboratory, this model has been used to identify cellular phenotypic changes at specific time point during this remodeling process10, 11. For example, class III &beta;-tubulin identifies pericytes along angiogenic vessels (Figure 6). In unstimulated tissues, class III &beta;-tubulin expression is nerve specific. In contrast, during the peak of capillary sprouting, class III &beta;-tubulin is expressed by perivascular cells. This type of result highlights the use of this simple and robust angiogenic model to identify novel cell types involved in the angiogenic process.
<img alt="Figure 1" src="/files/ftp_upload/3954/3954fig1.jpg" /> 
	Figure 1. Images of the plastic stage pre- and post-modification. The pre-modified stage is a 100 mm Petri dish. Modifications include an elliptical hole cut in the center and the subsequent addition of modeling clay or silicon glue to the hole's edge for the creation of a raised, smooth surface. This surface provides an inner boundary that facilitates superfusion of the exteriorized mesenteric windows. Scale bar is 1 cm.
<img alt="Figure 2" src="/files/ftp_upload/3954/3954fig2.jpg" /> 
	Figure 2. Image of exteriorized mesenteric region. Mesenteric windows are defined as the thin translucent membranes between the artery/vein pairs feeding the small intestine. During the exteriorization duration, the mesenteric region is laid out and immersed in saline inside a modified Petri dish. Inert yellow modeling clay provides a smooth surface for the mesentery to be pulled through the pre-cut hole. Scale bar is 1 cm.
<img alt="Figure 3" src="/files/ftp_upload/3954/3954fig3.jpg" /> 
	Figure 3. Representative images of mesenteric microvascular networks from unstimulated tissues and tissues at 3 and 10 days post exteriorization of the mesentery. PECAM labeling identified the hierarchy of microvascular networks including, arterioles (A), venules (V) and capillaries (C). Post stimulation, microvascular networks displayed an increase in capillary sprouting (arrow heads) and vessel density. Scale bar is 100 &mu;m.
<img alt="Figure 4" src="/files/ftp_upload/3954/3954fig4.jpg" /> 
	Figure 4. Representative images of arteriole/venule pairs within adult rat mesenteric microvascular networks. In both images, arterioles (A) can be differentiated from venules (V) based on a smaller relative diameter and elongated endothelial cell morphology. Scale bars are 20 &mu;m.
<img alt="Figure 5" src="/files/ftp_upload/3954/3954fig5.jpg" /> 
	Figure 5. Representative quantification of angiogenic metrics over the time course of microvascular growth post mesentery exteriorization. A) Vascular area per tissue area. B) Number of capillary sprouts per vascular area. C) Total vascular length per vascular area. *represents significant difference compared to unstimulated group. Statistical comparisons were made using a One-Way ANOVA followed by Dunn's test. (p &lt; 0.05). UN represents unstimulated.
<img alt="Figure 6" src="/files/ftp_upload/3954/3954fig6.jpg" /> 
	Figure 6. Representative fluorescent images of mesenteric microvascular networks from unstimulated tissues and tissues at 3 and 10 days post exteriorization of the mesentery. Immunofluorescent PECAM labeling (red) identifies endothelial cells, and class III &beta;-tubulin labeling (green) identifies nerves (arrow heads) and perivascular cells (arrows). Perivascular cells transiently upregulate class III &beta;-tubulin during capillary sprouting. In unstimulated microvascular networks, class III &beta;-tubulin is nerve specific and does not identify perivascular cells. 3 days post stimulation, class III &beta;-tubulin positively labels perivascular cells along microvessels. By day 10, class III &beta;-tubulin expression pattern begins to return to the unstimulated scenario. Scale bar is 50 &mu;m.
<img alt="Figure 7" src="/files/ftp_upload/3954/3954fig7.jpg" /> 
	Figure 7. Images supporting the feasibility of tracking pre-labeled locally applied cells during microvascular network growth stimulated by the mesentery exteriorization. Cells were superfused over mesenteric windows during the 20-minute exteriorization period. 1 day post-surgery, DiI labeled cells (red) were observed in the dame focal plane with PECAM positive microvessels (green). A, B) Examples of DiI labeled bone marrow cells exhibiting rounded and elongated morphologies. In some cases (arrows) cells were elongated along microvessels. C) Example of a cluster of DiI labeled mesenchymal stem cells near the tip of a capillary sprout (arrow). Scale bars are 50 &mu;m (A), and 20 &mu;m (B, C).

<ol>
	<li>Carmeliet, P., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+cancer+and+other+diseases.">Angiogenesis in cancer and other diseases.</a> Nature. 407, 249-257 (2000).</li><li>le Noble, F. A., Stassen, F. R., Hacking, W. J., Struijker Boudier, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+and+hypertension.">Angiogenesis and hypertension.</a> J. Hypertens. 16, 1563-1572 (1998).</li><li>Peirce, S. M., Skalak, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+remodeling:+a+complex+continuum+spanning+angiogenesis+to+arteriogenesis.">Microvascular remodeling: a complex continuum spanning angiogenesis to arteriogenesis.</a> Microcirculation. 10, 99-111 (2003).</li><li>Anderson, C. R., Ponce, A. M., Price, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+identification+of+an+extracellular+matrix+scaffold+that+microguides+capillary+sprouting+in+vivo.">Immunohistochemical identification of an extracellular matrix scaffold that microguides capillary sprouting in vivo.</a> J. Histochem. Cytochem. 52, 1063-1072 (2004).</li><li>Ponce, A. M., Price, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenic+stimulus+determines+the+positioning+of+pericytes+within+capillary+sprouts+in+vivo.">Angiogenic stimulus determines the positioning of pericytes within capillary sprouts in vivo.</a> Microvasc. Res. 65, 45-48 (2003).</li><li>Franzen, L., Ghassemifar, R., Malcherek, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+mast+cell+activation+improves+connective+tissue+repair+in+the+perforated+rat+mesentery.">Experimental mast cell activation improves connective tissue repair in the perforated rat mesentery.</a> Agents Actions. 33, 371-377 (1991).</li><li>Anderson, C. R., Hastings, N. E., Blackman, B. R., Price, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+sprout+endothelial+cells+exhibit+a+CD36+low+phenotype:+regulation+by+shear+stress+and+vascular+endothelial+growth+factor-induced+mechanism+for+attenuating+anti-proliferative+thrombospondin-1+signaling.">Capillary sprout endothelial cells exhibit a CD36 low phenotype: regulation by shear stress and vascular endothelial growth factor-induced mechanism for attenuating anti-proliferative thrombospondin-1 signaling.</a> Am. J. Pathol. 173, 1220-1228 (2008).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+models+of+angiogenesis.">In vivo models of angiogenesis.</a> J. Cell. Mol. Med. 10, 588-612 (2006).</li><li>Norrby, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+Mesentery+Angiogenesis+Assay.">Rat Mesentery Angiogenesis Assay.</a> J. Vis. Exp. (52), e3078 (2011).</li><li>Murfee, W. L., Rehorn, M. R., Peirce, S. M., Skalak, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+cells+along+venules+upregulate+NG2+expression+during+microvascular+remodeling.">Perivascular cells along venules upregulate NG2 expression during microvascular remodeling.</a> Microcirculation. 13, 261-273 (2006).</li><li>Stapor, C. P., Murfee, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+class+III+&#946;-tubulin+as+a+marker+of+angiogenic+perivascular+cells.">Identification of class III &#946;-tubulin as a marker of angiogenic perivascular cells.</a> Microvascular Research. , (2011).</li><li>Yang, M., Aragon, M., Murfee, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+Mesenteric+Microvascular+Networks+from+Spontaneously+Hypertensive+Versus+Normotensive+Rats.">Angiogenesis in Mesenteric Microvascular Networks from Spontaneously Hypertensive Versus Normotensive Rats.</a> Microcirculation. , (2011).</li><li>Robichaux, J. 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We have nothing to disclose.

The exteriorization model was reported in 2006 and is adapted from previous mechanical injury rat mesentery models of angiogenesis4-7 and produces similar results to well established i.p. injection models that take advantage of the rat mesentery9. The 20 minute exteriorization time was experimentally determined to produce a robust angiogenic response. While this time period could be varied, it does allow for local application of angiogenic inhibitors4 for mechanistic studies and direct application of exogenous cells for cell lineage studies. Feasibility of cell incorporation into remodeling mesenteric tissue is supported by preliminary studies in our laboratory using pre-labeled bone marrow cells and mesenchymal stem cells (Figure 7), and by the success of investigating the fate of human adipose-derived stromal cells injection i.p.14. In our laboratory, we have used this model to identify pericyte phenotypic changes over the time course of the angiogenic response10 and to assess the angiogenic potential during pathological conditions, such as hypertension12. The angiogenic response and cell phenotype changes associated with this model can also be observed in other rat mesenteric angiogenic models including chronic hypoxia exposure10, 11.
A limitation of the exteriorization model is that the exact triggering mechanisms of angiogenesis are unknown. Exteriorization of the mesentery has been linked to mast cell degranulation and increased histamine levels6, yet further investigation is required to gain more insight. The angiogenic stimulus is undoubtedly multi-factorial, producing a robust remodeling response across the hierarchy of a microvascular network. While the unknown mechanisms remain a major critique of this model, its reproducibility and simplicity make it attractive for identifying cellular dynamics involved in the inherently complex capillary sprouting process. The reproducibility of the model is supported by comparable angiogenic metrics over the time course of microvascular network growth across multiple rat strains (male Wistar and female Sprague-Dawley) in previously published studies from our laboratory10, 12. Since, the majority of adult rat mesenteric tissues are vascularized, the model also allows for multiple tissues to be examined per animal. Unfortunately, this model is not obviously applicable to genetic mouse models as mouse mesenteric windows have less native vascularization and, in our experience, commonly lack observable branching networks. Future applications include the investigation of vessel functionality during angiogenesis using intra-vital microscopy at specific time points and the investigation of related cellular dynamics involved in lymphangiogenesis and neurogenesis. Though the extent of native vascularization per mesenteric window seems to be roughly proportional with age, we have observed branching microvascular networks in male Wistar rats as young as 4-5 weeks old. These observations suggest that the exteriorization model could also be applied to compare the angiogenic differences across ages.

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Additional Comments</td>
		</tr>
		<tr>
			<td>Drape</td>
			<td>Cardinal Health</td>
			<td>4012</td>
			<td>12"x12" Bio-Shield Regular Sterilization Wraps</td>
		</tr>
		<tr>
			<td>Noyes Micro Scissor</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5677</td>
			<td>Noyes Micro Dissecting Spring Scissors; 
			Straight, Sharp-Blunt Points; 13mm Cutting Edge;0.25mm Tip Width, 4 1/2" Overall Length</td>
		</tr>
		<tr>
			<td>Graefe Forcep</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5135</td>
			<td>Micro Dissecting Forceps; Serrated; Slight Curve; 0.8mm Tip Width; 4" Length</td>
		</tr>
		<tr>
			<td>Graefe Forcep</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5130</td>
			<td>Micro Dissecting Forceps; Serrated, Straight; 0.8mm Tip Width; 4" Length</td>
		</tr>
		<tr>
			<td>4-0 suture</td>
			<td>ETHICON</td>
			<td>699G</td>
			<td>(1.5 metric) ETHILON Nylon Suture Black Monofilament</td>
		</tr>
		<tr>
			<td>5-0 suture</td>
			<td>ETHICON</td>
			<td>8556</td>
			<td>(1.0 metric) PROLENE Polyprolene Suture Blue Monofilament</td>
		</tr>
		<tr>
			<td>7-0 suture</td>
			<td>ETHICON</td>
			<td>1647G</td>
			<td>(0.5 metric) ETHILON Nylon Suture Black Monofilament</td>
		</tr>
		<tr>
			<td>Castroviejo Micro Needle Holder</td>
			<td>Fine Science Tools</td>
			<td>12060-02</td>
			<td>Tip Width:0.6mm 
			Clamping Length:5mm 
			Length:9cm 
			Straight tip</td>
		</tr>
		<tr>
			<td>Castroviejo Needle Holder</td>
			<td>Fine Science Tools</td>
			<td>12565-14</td>
			<td>Tip Shape:Straight 
			Tip Width:1.5mm 
			Clamping Length:10mm 
			Scissors:No 
			Lock:Yes 
			Length:14cm 
			Serrated:Yes</td>
		</tr>
		<tr>
			<td>Scalpel Handle</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-9843</td>
			<td>Scalpel Handle, #3; Solid; 4" Length</td>
		</tr>
		<tr>
			<td>Sterile Surgical Blade</td>
			<td>Cincinnati Surgical</td>
			<td>0110</td>
			<td>Stainless Steel; Size 10</td>
		</tr>
		<tr>
			<td>Petri Dish</td>
			<td>Fisher Scientific</td>
			<td>08-757-13</td>
			<td>Beveled Ridge, Slippable</td>
		</tr>
	</tbody>
</table>
Table of Specific Surgical Materials and Tools.
<table border="1">
	<tbody>
		<tr>
			<td>Name </td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Additional Comments</td>
		</tr>
		<tr>
			<td>Beuthanasia</td>
			<td>Schering-Plough Animal Health Corp. Union (Ordered from MWI Veterinary Supply)</td>
			<td>MWI #: 011168</td>
			<td>Active Ingredient: Per 100mL, 390 mg pentobarbital sodium, 50mg phenytoin sodium</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Fort Dodge Animal Health (Ordered from MWI Veterinary Supply)</td>
			<td>MWI #: 000680</td>
			<td>Kateset 100 mg/ml</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>LLOYD. Inc. 
			(Ordered from MWI Veterinary Supply)</td>
			<td>MWI #: 009307</td>
			<td>Anased 100 mg/ml</td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Hospira Inc.</td>
			<td>94-217-JT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>SIGMA</td>
			<td>011M8207</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma</td>
			<td>BCBB4080</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PECAM (CD31)</td>
			<td>BD Pharmingen</td>
			<td>553371</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Streptavidin-CY3</td>
			<td>Jackson ImmunoResearch</td>
			<td>016-160-084</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Jackson ImmunoResearch</td>
			<td>096555</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>VECTASTAIN Elite ABC</td>
			<td>Vector Laboratories</td>
			<td>PK-6100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vector Nova Red</td>
			<td>Vector Laboratories</td>
			<td>SK-4800</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>VectaMount</td>
			<td>Vector Laboratories</td>
			<td>H-500</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Table of Specific Reagents

rat mesentery exteriorization: a model for investigating the cellular dynamics involved in angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

Watch this Scientific Journal Video about Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis at JoVE.com

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

rat-mesentery-exteriorization-model-for-investigating-cellular

Research

JoVE Journal

Biology

19.0K Views.  Tulane University. This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

Video: Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.