We are grateful to all the members of the Dunnwald Lab who have contributed to the optimization of this protocol over the years, and to Gina Schatteman whose persistence in promoting the use of serial sectioning for wound analysis made its creation possible. This work was supported by funding from NIH/NIAMS to Martine Dunnwald (AR067739).

All experiments were completed in accordance and compliance with federal regulations and University of Iowa policy and procedures have been approved by the University of Iowa IACUC.
1. Animals and husbandry
<ol>
	<li>Use adult mice of the desired mouse line at 8-10 weeks of age when the hair follicle stage is in telogen.</li>
	<li>On the day of surgery, separate mice into clean cages and individually house to minimize wound disruption.</li>
</ol>
2. Surgery
NOTE: It is unnecessary to maintain sterile surgical conditions. While care should be taken to maintain sterility between animals, the punch biopsy itself is done on a clean, but nonsterile surface. The surgery duration per animal is between 10 and 15 min.
<ol>
	<li>Anesthetization
	<ol>
		<li>Anesthetize the animal for 1-2 min in an induction chamber with the isoflurane vaporizer set to a 4-5% flow rate and the oxygen flow meter set at 1 liter per minute. See Discussion for alternative anesthesia options.</li>
		<li>Confirm proper anesthetization before beginning the procedure. The depth of anesthesia can be confirmed by a firm toe pinch.</li>
		<li>Transfer the mouse from the induction container to a nose cone and reduce the isoflurane flow rate to 1.5% and the oxygen flow meter to 0.5 L/min.</li>
		<li>Apply ophthalmic ointment to both eyes as the procedure exceeds 5 min.</li>
		<li>Maintain normal body temperature using a thermal pad.</li>
	</ol>
	</li>
	<li>Preparation of the wound site
	<ol>
		<li>Use an electric razor clipper in a caudal rostral motion to remove the fur on the back of the mouse at the shoulder level. Remove hair lower on the back as needed if performing more than two wounds.</li>
		<li>Remove the remaining hair by using a razor blade in a rostral caudal motion held at 20&#730; from the back of the mouse to closely shave the clipped area (Figure 1A).</li>
		<li>Clean the shaved area with a povidone-iodine swab.</li>
		<li>Wipe the skin with a sterile 70% isopropyl alcohol prep pad to reduce potential cutaneous irritation from the iodine swab.</li>
	</ol>
	</li>
	<li>Wounding
	<ol>
		<li>Pinch the skin between the shoulder blades along the dorsal midline and pull the sandwiched skinfold away from the body (Figure 1B).</li>
		<li>Position the mouse on its side with the skinfold on a flat surface draped with a clean paper-based towel or equivalent. Use a sheet of dental wax underneath the towel to protect the underlying surface from damage (Figure 1C).</li>
		<li>Place the biopsy punch of desired size as close to the body as possible and allow the skin to relax. Do not stretch the skin, or the wound size will be larger than the designated punch size (Figure 1D).</li>
		<li>Punch the skin by pressing down, a rocking motion may be used to ensure all layers of the skin on both sides have been penetrated (Figure 1E). Use a new biopsy punch for each animal.</li>
		<li>Remove the punch biopsies from the wounds (Figure 1F). If there are still sites of attachment use sterile scissors and tweezers to free the punch from the surrounding skin. Process the punch biopsy control tissue as required based on downstream plans for wound healing analyses (see Discussion for suggestions).</li>
		<li>Take macroscopic photographs from an equal distance to the wound sites or with a ruler in the frame in order to measure the initial wound area and eliminate outliers from analysis.</li>
		<li>Administer analgesia for a minimum of 24 h in accordance with an approved animal protocol. For example: Buprenorphine SR-LAB, injected as a single dose subcutaneously at 0.5-2 mg/kg for 48 h of pain relief (see Discussion for alternate suggestions and considerations).</li>
		<li>Monitor the mouse as it comes out of anesthesia until it maintains an upright posture and is walking normally around the cage.</li>
	</ol>
	</li>
</ol>
3. Post wound monitoring
<ol>
	<li>Monitor mice daily for experimental endpoints as determined by the investigator and in accordance and compliance with institutional protocols. Examples include: infection, visible weight loss, or a hunched posture.</li>
	<li>Take daily macroscopic photographs in a controlled manner as was done after the initial surgery.</li>
</ol>
4. Harvesting wounds
<ol>
	<li>Euthanize mice at the desired time point post-wounding in accordance with an approved animal protocol.</li>
	<li>Take macroscopic photographs of the wound sites in a controlled manner consistent with previous photograph acquisition (Figure 2A,C).</li>
	<li>Cut a wide rectangle around the wound sites using a scalpel (Figure 2D,E).</li>
	<li>Free the rectangular piece of tissue using scissors and tweezers to peel back and cut the skin away from the underlying tissue (Figure 2F).&#160;&#160;Place in a Petri dish (Figure 2G).</li>
	<li>Harvest the wounds. Trim down to 2 mm of unwounded tissue surrounding all sides of the wound in a rectangular shape&#160;(Figure 2H,I). See Discussion for alternative options to harvest the wound.</li>
	<li>Process the wounds as required for subsequent studies. Reserve at least one wound per mouse for paraffin embedding and histological analysis.</li>
</ol>
5. Wound fixation and embedding
<ol>
	<li>Fix the wound
	<ol>
		<li>Fix the wound tissue in a freshly prepared 4% paraformaldehyde solution8 for 3 h at room temperature then transfer to 4 &#730;C overnight. Electron microscopy (EM) grade paraformaldehyde and solution filtration is not required.</li>
		<li>Wash the wounds twice for 30 min in 1x PBS.</li>
		<li>Replace PBS with 70% ETOH and store at 4 &#730;C until embedding. Process tissues within 24-48 h to avoid antigen loss or within 1-2 weeks if only evaluating histological characteristics.</li>
	</ol>
	</li>
	<li>Process and embed the wound
	<ol>
		<li>Transfer each wound to an embedding cassette. Label embedding cassettes in pencil as the process will remove inks.</li>
		<li>Process the tissue either manually or using an automated processor by dehydrating the tissue with increasing ethanol percentages, clearing with xylene, and then infiltrating the tissue with paraffin wax (Table 1).</li>
		<li>Embed the wound 90&#730; (&#8220;standing&#8221;) from the horizontal surface of the embedding mold (Figure 3A,B).</li>
	</ol>
	</li>
</ol>
6. Day 0 wound area analysis
<ol>
	<li>Download NIH-Image J or NIH-Fiji free software (https://imagej.net/Fiji/Downloads).</li>
	<li>Open a file with a photograph of day 0 wounds.</li>
	<li>Check the box for &#8220;Area&#8221; under Analyze | Set Measurements.</li>
	<li>Select &#8220;Set Scale&#8221; under Analyze. Enter the distance in pixels, the known corresponding distance and the unit of the distance (= unit of length) if macroscopic measurements are part of the study or skip this step if only relative measurements are required.</li>
	<li>Select &#8220;Freehand selections&#8221; on the Fiji toolbar.</li>
	<li>Outline the perimeter of the wound.</li>
	<li>Click Measure under Analyze.</li>
	<li>Create a spreadsheet to keep track of the measurements per animal per wound.</li>
	<li>Copy the measurement of the wound area in the spreadsheet.</li>
	<li>Calculate the mean area and the standard deviation of all wounds for a given experiment.</li>
	<li>Exclude any wounds outside two standard deviations of the mean from histological analysis.</li>
</ol>
7. Serial sectioning
<ol>
	<li>Chill the paraffin-embedded wound blocks at 4 &#730;C overnight.</li>
	<li>Insert the paraffin block on the block holder of the microtome and orient so the blade will cut straight across the block. Orient the block such that the tissue &#8220;stands&#8221; at 90&#730; allowing the simultaneous sectioning of the epidermis and dermis (Figure 3C,D).</li>
	<li>Make 2-4 ribbons of 20-30 paraffin sections of 7 &#181;m each.</li>
	<li>Use a dry paint brush and a dissection teasing needle to transfer each ribbon to a firm yet manipulatable surface such as a firm black plastic sheet.</li>
	<li>Detach the top section of each ribbon with a razor blade and place on a microscope slide.</li>
	<li>Observe the unstained sections under a brightfield microscope to determine which ones contain wounded tissue, which can be identified by absence of hair follicle, changes in the appearance of the connective tissue or the epidermis, and/or the presence of a scab (Figure 4A,B).</li>
	<li>Discard unwounded sections up to 20 sections before the beginning of the wound.</li>
	<li>Section through the wound by repeating steps 7.3 and 7.4 until no wound is detected in unstained sections.</li>
</ol>
8. Mounting of paraffin sections
<ol>
	<li>Separate paraffin sections every 5 sections with a razor blade, starting with the first ribbon (Figure 3E).</li>
	<li>Label microscope slides with both the slide number and all the section numbers.</li>
	<li>Grab the group of 5 sections with a wet paint brush and float them on the surface of the water of a warm water bath (40-45 &#730;C) to flatten them out.</li>
	<li>Pick the group of 5 sections out of the water bath using one of the labelled microscope slides (Figure 3F) and place on a slide warmer set at 37 &#730;C for up to 24 h.</li>
	<li>Store the slides upright in a slide box.</li>
</ol>
9. Histological staining
<ol>
	<li>Transfer every 8th microscope slide (equivalent to every 40th paraffin section) to a staining rack and stain with hematoxylin and eosin.</li>
</ol>
10. Microscopic imaging
<ol>
	<li>Acquire images using a bright field microscope equipped with a 4x objective and digital acquisition capabilities. Record the scale at which the image is taken.</li>
	<li>Image the entire wound of the top section of each stained slide and make sure to include some unwounded tissue on either side. Take multiple overlapping pictures if the wound is larger than the frame of a single picture.</li>
	<li>Save the file including the section number for morphometric analysis. Use the section number followed by a, b, c, etc. for overlapping pictures of the same wound.</li>
</ol>
11. Morphometric analysis
NOTE: When the wound spans multiple pictures, sum the measurements taken from the individual pictures to obtain one value per metric per wound section to record in the spreadsheet.
<ol>
	<li>In Image J, open a digital file of a stained wound picture. Do not use stitched pictures for analysis. Perform measurements on zoomed-in overlapping pictures by finding landmarks to leave off and pick up measurements from picture to picture.</li>
	<li>Set the scale and measurement preferences.
	<ol>
		<li>Select &#8220;Set Scale&#8221; under Analyze. Enter the distance in pixels, the known corresponding distance and the unit of the distance (= unit of length). The scale should appear in the window and should correspond to the scale the image was acquired at.</li>
		<li>Check the box &#8220;Global&#8221; to keep the scale the same for each open image.</li>
		<li>Repeat steps 11.2.1 to 11.2.2 every time Image J is closed and reopened.</li>
		<li>Check the box for &#8220;Area&#8221; under Analyze | Set Measurements.</li>
	</ol>
	</li>
	<li>Measure the wound length
	<ol>
		<li>Select &#8220;Freehand selections&#8221; on the Fiji toolbar.</li>
		<li>Measure starting from the last hair follicle of the uninjured tissue on one side of the wound to the first hair follicle of the uninjured tissue on the other side of the wound (Figure 4A,B).</li>
		<li>Trace along the dermo-epidermal junction to reach these two landmarks. If the epidermis does not cover the entire wound, follow the dermo-epidermal junction on one side of the wound and where the migrating tongue ends continue following the superior aspect of the granulation tissue or the junction between the granulation tissue and the scab until you reach the migrating tongue and then finally the first hair follicle of the uninjured tissue on the other side (Figure 4E,F).</li>
		<li>Under Analyze, click Measure. The length of the measurement will appear in the same units as set in the scale.</li>
		<li>Create a spreadsheet to keep track of the measurements (Supplementary Table 1).</li>
		<li>Copy the wound length into the spreadsheet.</li>
	</ol>
	</li>
	<li>Measure the epidermal length
	<ol>
		<li>If the epidermis covers the entire wound, the epidermal length is the same as the wound length.
		<ol>
			<li>Copy the &#8220;wound length&#8221; measurement into the &#8220;epidermal length&#8221; column in the Excel spreadsheet and skip to step 11.5.</li>
		</ol>
		</li>
		<li>If the epidermis does not cover the entire wound, select the &#8220;Freehand selections&#8221; and measure the distance between each epidermal leading edge following the superior aspect of the granulation tissue or the junction between the granulation tissue and the scab to the first hair follicle (Figure 4C,D).
		<ol>
			<li>Under Analyze, click Measure.</li>
			<li>Subtract this measurement from the wound length and record the number under &#8220;epidermal length&#8221; in the Excel spreadsheet.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Measure the wound area
	<ol>
		<li>Select &#8220;Freehand selections&#8221; on the Fiji toolbar.</li>
		<li>Measure starting from the last hair follicle of the uninjured tissue on one side of the wound to the first hair follicle of the uninjured tissue on the other side of the wound (Figure 4A,B,E,F).</li>
		<li>Trace along the superior aspect of the epidermis (do not include the scab) or the superior aspect of the granulation tissue if the wound is not fully covered by the epidermis.</li>
		<li>Continue to trace vertically along the hair follicle into the granulation tissue once the opposite hair follicle is reached and until adipose tissue or muscle is reached. Follow the inferior border of the granulation tissue to the opposite side of the wound and join the starting point along the hair follicle to close the area (Figure 4E,F).</li>
		<li>Under Analyze, click Measure.</li>
		<li>Copy the wound area into the spreadsheet under &#8220;wound area measured.&#8221;</li>
	</ol>
	</li>
	<li>Measure the epidermal area
	<ol>
		<li>Select &#8220;Freehand selections&#8221; on the Fiji toolbar.</li>
		<li>If the wound is fully epithelialized:
		<ol>
			<li>Trace along the superior aspect of the epidermis until the opposite hair follicle is reached and complete the area by &#8220;returning&#8221; to the starting point following the dermo-epidermal junction between the epidermis and dermis (Figure 4D).</li>
			<li>Under Analyze, click Measure.</li>
			<li>Copy the epidermal area into the Excel spreadsheet under &#8220;epidermal area measured&#8221; and skip to step 11.7.</li>
		</ol>
		</li>
		<li>If the wound is not fully epithelialized:
		<ol>
			<li>Trace along the superior aspect of the epidermis until the leading edge and return to the starting point following the dermo-epidermal junction (Figure 4C).</li>
			<li>Under Analyze, click Measure.</li>
			<li>Repeat step 11.6.3.1 and 11.6.3.2 on the opposite side of the wound.</li>
			<li>Under Analyze, click Measure.</li>
			<li>Sum the two numbers obtained in steps 11.6.3.2 and 11.6.3.4 and enter the result under &#8220;epidermal area measured&#8221; in the spreadsheet.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Repeat steps 11.3 to 11.6 on every 40th section&#160;(every 8th slide).</li>
	<li>Calculate the epidermal area of the entire wound.
	<ol>
		<li>Create a new column in the spreadsheet &#8220;epidermal area calculated&#8221; next to &#8220;epidermal length.&#8221;</li>
		<li>Multiply the number for &#8220;epidermal length&#8221; by 280 for each section except the last one (7 &#181;m thick section x 40 sections).</li>
		<li>Multiply the number for &#8220;epidermal length&#8221; by 7 for the last section (thickness of the section).</li>
		<li>Sum the value of the &#8220;epidermal area calculated&#8221; for each section to obtain the epidermal area of the entire wound.</li>
	</ol>
	</li>
	<li>Calculate the wound area of the entire wound.
	<ol>
		<li>Repeat steps 11.8.1 to 11.8.4 using the wound length measurements.</li>
	</ol>
	</li>
	<li>Calculate the epidermal volume of the entire wound.
	<ol>
		<li>Repeat steps 11.8.1 to 11.8.4 using the &#8220;epidermal area measured&#8221; measurements.</li>
	</ol>
	</li>
	<li>Calculate the wound volume of the entire wound.
	<ol>
		<li>Repeat steps 11.8.1 to 11.8.4 using the &#8220;wound area measured&#8221; measurements.</li>
	</ol>
	</li>
	<li>Calculate the percentage of epidermal volume in the wound.
	<ol>
		<li>Divide the total epidermal volume by the total wound volume and multiply by 100 to obtain the percentage (do not do this ratio for each section).</li>
	</ol>
	</li>
	<li>Calculate the percentage of epidermal area among the wound area.
	<ol>
		<li>Divide the total epidermal area by the total wound area and multiply by 100 to obtain the percentage. If a wound is fully epithelialized, this number should be 100.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

The bilateral excisional wound model is a highly customizable procedure which can be used to study many different aspects of wound healing. Before beginning a wound healing project, investigators should perform a power analysis to determine the number of wounds needed to detect a defect of a particular effect size. Inconsistencies exist within the literature on whether individual mice or wounds should be used as biological replicates, however, a recent study showed that there is no significant correlation between two wou...

Cutaneous wound healing is a complex biological process with sequentially overlapping phases. It requires the coordination of cellular and molecular processes that are temporally and spatially regulated in order to restore the barrier function of the damaged epithelium. In the first phase, inflammation, neutrophils and macrophages migrate into the wound, mobilizing local and systemic defenses1. Following and overlapping the inflammatory phase is the proliferation stage. Fibroblasts begin rapidly proliferating and migrating into the granulation tissue. Keratinocytes away from the leading edge directionally proliferate towards the wound as differentiated keratinocytes in the leading edge migrate to re-epithelialize the wound2. Finally, the remodeling and maturation phase begins, during which fibroblasts in the granulation tissue start to synthesize and deposit collagen. The remodeling and organization of the new matrix can last up to 1 year following injury3. Due to the complexity of overlapping events involving cross-talk between multiple cell types, and despite years of research, many of the cellular and molecular mechanisms underlying wound healing remain poorly understood.
The mouse model is the predominant mammalian model for investigating mechanisms of wound healing due to their ease of use, relatively low cost and genetic manipulability1,4,5. Although different types of wounds have been described in the murine model, the most common is an excisional wound (either bilateral punch or direct punch biopsy), followed by incisional wound models4. The excisional wound model has a distinct advantage over the incisional model as it inherently generates control tissue that has not undergone the healing process. The punch biopsy tissue that is excised as part of the surgical protocol can be processed in the same manner as the wounded tissue and used to establish the homeostatic conditions for a desired criterion. Excised control tissue may also be useful if assessing the effects of a skin pretreatment or confirming successful gene alteration at the time of injury4.
Healing parameters can be assessed by many different techniques, including planimetry or histology. However, planimetry can only evaluate visible characteristics of the wound, and due to the presence of a scab, often does not correlate to measurements of healing that are visualized by histology, thereby making histology the &#8220;gold standard&#8221; of analysis4. Despite histological analysis being the gold standard, it is most often performed on an arbitrary subset of the wound6,7. For instance, cutting the wound in &#8220;half&#8221; prior to embedding and sectioning the wound is currently common practice to reduce the time and resources spent on sectioning materials and data analysis. The method of morphometric analysis described in this protocol was developed to encompass the entire wound tissue, to accurately reflect the morphological characteristics of the wound, and to increase the likelihood of detecting wound healing defects with a small effect size. In this protocol, we detail a surgical method for generating the most commonly studied murine wound, the bilateral full-thickness excisional wound, as well as a detailed and rigorous method for histological analysis such is rarely used in the field.

<table>
	<tbody>
		<tr>
			<td>100% ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>80% ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>95% ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol Prep</td>
			<td>NOVAPLUS</td>
			<td>V9100</td>
			<td>70% Isopropyl alcohol, sterile</td>
		</tr>
		<tr>
			<td>Ammonium hydroxide</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy pads</td>
			<td>Cellpath</td>
			<td>22-222-012</td>
			<td></td>
		</tr>
		<tr>
			<td>Black plastic sheet</td>
			<td></td>
			<td></td>
			<td>Something firm yet manipulatable about the size of a sheet of paper</td>
		</tr>
		<tr>
			<td>Brightfield microscope</td>
			<td></td>
			<td></td>
			<td>With digital acquisition capabilities and a 4X objective</td>
		</tr>
		<tr>
			<td>Cotton tipped applicators</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td></td>
			<td></td>
			<td>22 x 60 #1</td>
		</tr>
		<tr>
			<td>Dental wax sheets</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digital camera</td>
			<td></td>
			<td></td>
			<td>Include a ruler for scale, if applicable</td>
		</tr>
		<tr>
			<td>Dissection teasing needle (straight)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding molds</td>
			<td></td>
			<td></td>
			<td>22 x 22 x 12</td>
		</tr>
		<tr>
			<td>Embedding rings</td>
			<td>Simport Scientific Inc.</td>
			<td>M460</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hair clipper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Conair</td>
			<td></td>
			<td>Moist dry Heating Pad</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome blades</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paint brushes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin Type 6</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Permount</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer solution (PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-iodine</td>
			<td>Aplicare</td>
			<td>82-255</td>
			<td></td>
		</tr>
		<tr>
			<td>Processing cassette</td>
			<td>Simport Scientific Inc.</td>
			<td>M490-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>ASR</td>
			<td></td>
			<td>.009 Regular Duty</td>
		</tr>
		<tr>
			<td>Scalpel blades #10</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sharp surgical scissors</td>
			<td></td>
			<td></td>
			<td>sterile for surgery</td>
		</tr>
		<tr>
			<td>Skin biopsy punches</td>
			<td></td>
			<td></td>
			<td>Size as determined by researcher</td>
		</tr>
		<tr>
			<td>Slide boxes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Slide warmers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrosted microscope slides</td>
			<td>Fisher Scientific</td>
			<td>22 037 246</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature control water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue embedding station</td>
			<td></td>
			<td></td>
			<td>Minimum of a paraffin dispenser and a cold plate</td>
		</tr>
		<tr>
			<td>Tissue processor</td>
			<td></td>
			<td></td>
			<td>Minimum of a oven with a vacuum pump</td>
		</tr>
		<tr>
			<td>Triple antibiotic opthalmic ointment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tweezers, curved tip</td>
			<td></td>
			<td></td>
			<td>sterile for surgery</td>
		</tr>
		<tr>
			<td>tweezers, tapered tip</td>
			<td></td>
			<td></td>
			<td>sterile for surgery</td>
		</tr>
		<tr>
			<td>WypAll X60</td>
			<td>Kimberly-Clark</td>
			<td>34865</td>
			<td></td>
		</tr>
	</tbody>
</table>

murine excisional wound healing model and histological morphometric wound analysis

The murine excisional wound model has been used extensively to study each of the sequentially overlapping phases of wound healing: inflammation, proliferation and remodeling. Murine wounds have a histologically well-defined and easily recognizable wound bed over which these different phases of the healing process are measurable. Within the field, it is common to use an arbitrarily defined &#8220;middle&#8221; of the wound for histological analyses. However, wounds are a three-dimensional entity and often not histologically symmetrical, supporting the need for a well-defined and robust method of quantification to detect morphometric defects with a small effect size. In this protocol, we describe the procedure for creating bilateral, full-thickness excisional wounds in mice as well as a detailed instruction on how to measure morphometric parameters using an image processing program on select serial sections. The two-dimension measurements of wound length, epidermal length, epidermal area, and wound area are used in combination with the known distance between sections to extrapolate the three-dimension epidermal area covering the wound, overall wound area, epidermal volume and wound volume. Although this detailed histological analysis is more time and resource consuming than conventional analyses, its rigor increases the likelihood of detecting novel phenotypes in an inherently complex wound healing process.

Figure 5 depicts the range in measured and calculated values obtained by performing morphometric analysis on wild-type wounds generated in different mouse strains by multiple surgeons and analyzed by different individuals. Wild-type mice from different strains can display statistical differences as described both in our studies and in the literature9,10. Based on these representative results, we recommend that, within one study, mice...

Watch this Scientific Journal Video about Murine Excisional Wound Healing Model and Histological Morphometric Wound Analysis at JoVE.com

Murine Excisional Wound Healing Model and Histological Morphometric Wound Analysis

This protocol describes how to generate bilateral, full-thickness excisional wounds in mice and how to subsequently monitor, harvest, and prepare the wounds for morphometric analysis. Included is an in-depth description of how to use serial histological sections to define, precisely quantify and detect morphometric defects.

The murine excisional wound model has been used extensively to study each of the sequentially overlapping phases of wound healing: inflammation, ...

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Developmental Biology

11.6K Views.  University of Iowa. This protocol describes how to generate bilateral, full-thickness excisional wounds in mice and how to subsequently monitor, harvest, and prepare the wounds for morphometric analysis. Included is an in-depth description of how to use serial histological sections to define, precisely quantify and detect morphometric defects.

Video: Murine Excisional Wound Healing Model and Histological Morphometric Wound Analysis

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell migration. However, when explants are established, these cells also move collectively, maintaining many of the features which make individual keratocytes an attractive model to study migration: rapid rates of motility, extensive actin-rich lamellae with a perpendicular actin cable, and relatively constant speed and direction of migration. In early explants, the rapid interconversion of cells migrating individually with those migrating collectively allows the study of the role of cell-cell adhesions in determining the mode of migration, and emphasizes the molecular links between the two modes of migration. Cells in later explants lose their ability to migrate rapidly and collectively as an epithelial to mesenchymal transition occurs and genes associated with wound healing and inflammation are differentially expressed. Thus, keratocyte explants can serve as an in vitro model for the reepithelialization that occurs during cutaneous wound healing and can represent a unique system to study mechanisms of collective cell migration in the context of a defined program of gene expression changes. A variety of mutant and transgenic zebrafish lines are available, which allows explants to be established from fish with different genetic backgrounds. This allows the role of different proteins within these processes to be uniquely addressed. The protocols outlined here describe an easy and effective method for establishing these explant cultures for use in a variety of assays related to collective cell migration.

Zebrafish keratocytes migrate in cell sheets from explants and provide an in vitro model for the study of the mechanisms of collective cell migration in the context of epithelial wound healing. These protocols detail an effective way to establish primary explant cultures for use in collective cell migration assays.

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell ...

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

Automated Quantification of Hematopoietic Cell &amp;#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and inflammatory responses. Given the current interest in the role of mesenchymal stromal cells in mediating tissue repair, we are interested in quantifying the migratory capacity of these cells, and understanding how migratory capacity may be altered after damage. Optimization of a rigorously quantitative migration assay that is both easy to customize and cost-effective to perform is key to answering questions concerning alterations in cell migration in response to various stimuli. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays, possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness. Despite its prominent use, the scratch assay is confounded by issues with reproducibility related to damage of the cell microenvironment, impediments to cell migration, influence of neighboring senescent cells, and cell proliferation, as well as lack of rigorous quantification. The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.

The capacity to migrate is a key function of many different cell types, including mesenchymal stromal cells (MSCs). However, quantifying alterations in migratory capacity after damage is challenging. This protocol describes an easily adaptable migration assay that uses rigorous statistics to quantify changes in MSC migratory capacity after damage.

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and ...

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor metastasis. In this assay, cells are grown to form a confluent monolayer and a mechanical wound is created by scratching with a device. Then the migration rate of the cells towards the denuded area can be monitored by imaging. Our 8-channel mechanical wounder is designed to tackle most of the problems associated with the cell migration assay. Firstly, our wounder can be easily sterilized by autoclaving or with common disinfectants. Secondly, the individual adjustable pins allow even contact with the cell culture plate so that sharp and reproducible wounds can be created. Thirdly, the guiding bars on both sides of the wounder ensure consistent wounding position in each well. The use of disposable plastic pipette tips for wounding can further provide better handling of the wounder as well as to minimize cross-contamination. In conclusion, our cell wounder can provide researchers with a user friendly and reproducible device for performing the cell migration assay using the standard 96-well culture plate.

Here, we present a protocol to perform a semi-high throughput cell migration assay on a 96-well cell culture plate. This protocol is a fast, simple and economical method to create consistent scratch wounds on a cell monolayer.

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related morbidity and mortality and affects more than 2 million people in the United States. A better understanding of the cellular and molecular mechanisms underlying alcohol-induced liver injury is crucial for developing effective treatment for ALD. Zebrafish larvae exhibit hepatic steatosis and fibrogenesis after just 24 h of exposure to 2% ethanol, making them useful for the study of acute alcoholic liver injury. This work describes the procedure for acute ethanol treatment in zebrafish larvae and shows that it causes steatosis and swelling of the hepatic blood vessels. A detailed protocol for Hematoxylin and Eosin (H&#38;E) staining that is optimized for the histological analysis of the zebrafish larval liver, is also described. H&#38;E staining has several unique advantages over immunofluorescence, as it marks all liver cells and extracellular components simultaneously and can readily detect hepatic injury, such as steatosis and fibrosis. Given the increasing usage of zebrafish in modeling toxin and virus-induced liver injury, as well as inherited liver diseases, this protocol serves as a reference for the histological analyses performed in all these studies.

This protocol describes histological analyses of the livers from zebrafish larvae that have been treated with 2% ethanol for 24 h. Such acute ethanol treatment results in hepatic steatosis and swelling of the hepatic vasculature.

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related ...

Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These interactions are provided by junctional proteins that form nexuses between adjacent cells. Junctional protein mislocalization and reduced physical associations with their binding partners can result in the loss of function and, consequently, to organ dysfunction. Thus, identifying protein localization and protein-protein interactions (PPIs) in normal and disease-related tissues is essential to finding new evidences and mechanisms leading to the progression of diseases or alterations in developmental status. This manuscript presents a two-step method to evaluate PPIs in murine mammary glands. In protocol section 1, a method to perform co-immunofluorescence (co-IF) using antibodies raised against the proteins of interest, followed by secondary antibodies labeled with fluorochromes, is described. Although co-IF allows for the demonstration of the proximity of the proteins, it does make it possible to study their physical interactions. Therefore, a detailed protocol for co-immunoprecipitation (co-IP) is provided in protocol section 2. This method is used to determine the physical interactions between proteins, without confirming whether these interactions are direct or indirect. In the last few years, co-IF and co-IP techniques have demonstrated that certain components of intercellular junctions co-localize and interact together, creating stage-dependent junctional nexuses that vary during mammary gland development.

Intercellular junctions are requisites for mammary gland stage-specific functions and development. This manuscript provides a detailed protocol for the study of protein-protein interactions (PPIs) and co-localization using murine mammary glands. These techniques allow for the investigation of the dynamics of the physical association between intercellular junctions at different developmental stages.

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These ...

Methods Article

Murine Excisional Wound Healing Model and Histological Morphometric Wound Analysis