We would like to thank Mr Paolo Matteuci and Mr George Smith for providing patient tissue. We are also grateful to Miss Amber Rose Stafford for assisting with tissue collection and the Daisy Appeal for providing laboratory facilities.

Human skin was obtained from patients undergoing reconstructive surgery at Castle Hill Hospital and Hull Royal Infirmary (Hull, UK) under full informed, written patient consent, institutional guidelines, and ethical approval (LRECs: 17/SC/0220 and 19/NE/0150). Non-diabetic skin was collected from patients undergoing routine surgery (mean age = 68). Diabetic skin was selected from donors who had established type II diabetes and a history of ulceration (mean age = 81). Samples from surgery were transported in holding media and processed immediately upon arrival at the laboratory. All experimental steps using unfixed human tissue were performed at Biosafety Level-2 (BSL-2) in a class II laminar flow biosafety cabinet.
1. Preparation of skin culture media and staining reagents
NOTE: All reagent and consumable details are provided in the Table of Materials. Ensure all reagents and equipment used for the processing and culture of human tissue are sterile. Sterilize instruments prior to the use and decontaminate with disinfectant following contact with the tissue. Decontaminate waste products in 1% disinfectant before disposal.
<ol>
	<li>Holding media: Supplement high glucose Dulbecco&#39;s Modified Eagle Medium (DMEM) with 2 mM L-glutamine and 4% (v/v) antibiotic-antimycotic solution.</li>
	<li>Hank&#39;s balanced salt solution (HBSS) with antibiotics: Add 4% (v/v) antibiotic-antimycotic solution to HBSS. Store at 4 &#176;C until use.</li>
	<li>Dulbecco&#39;s phosphate buffered saline (DPBS): Prepare DPBS by dissolving 9.6 g of DPBS powder per liter of distilled water (dH2O). Autoclave to sterilize and store at 4 &#176;C until use.</li>
	<li>Human skin growth media: Supplement high glucose DMEM with 2 mM L-glutamine, 1% (v/v) antibiotic-antimycotic solution and 10% (v/v) fetal bovine serum. Store at 4 &#176;C until use.</li>
	<li>Skin fixative: To 450 mL of dH2O, add 40 mL of formaldehyde solution, 10 mL of glacial acetic acid, 4.5 g of sodium chloride and 0.25 g of alkyltrimethylammonium bromide. Store at room temperature (RT) and use within a few days. 
	CAUTION: Fixative is hazardous (irritant and flammable). Handle with care and dispose of via an appropriate route.</li>
	<li>Phosphate buffered saline (PBS): Prepare PBS for whole-mount staining by adding 6 g of sodium chloride to 100 mL of phosphate buffer solution and 900 mL of dH2O.</li>
	<li>Staining wash buffer: Dissolve 0.5% (v/v) Triton X-100 in PBS.</li>
	<li>Blocking buffer: Add 0.2% (w/v) sodium azide and 2% (v/v) animal serum to staining wash buffer. Store at 4 &#176;C for up to two weeks. 
	NOTE: Block in the serum of the secondary antibody host species. Sodium azide will prevent bacterial growth during incubation.</li>
	<li>DAPI working solution: Prepare a 5 mg/mL stock of 4&#8242;,6-diamidino-2-phenylindole (DAPI) in dimethyl sulfoxide. Dilute the stock 1:1,000 in staining wash buffer to give a 5 &#181;g/mL DAPI working solution.</li>
	<li>Peroxidase block: Add 0.3% (v/v) hydrogen peroxide to staining wash buffer. Store at 4 &#176;C until use. Keep in the dark to prevent decomposition.</li>
	<li>ABC-HRP Kit:
	<ol>
		<li>HRP-conjugated secondary antibody: 1 drop of biotinylated rabbit anti-goat IgG in 5 mL of staining buffer. Store at 4 &#176;C for up to two weeks. 
		&#8203;NOTE: The kit/secondary used will depend on the host species of the primary antibody.</li>
		<li>Avidin-biotin complex (ABC) reagent: 2 drops of reagent A and 2 drops of reagent B in 5 mL of staining wash buffer. ABC reagent should be prepared at least 30 min prior to use. Store at 4 &#176;C for up to two weeks.</li>
	</ol>
	</li>
	<li>Peroxidase substrate: 3 drops of reagent 1, 2 drops of reagent 2, 2 drops of reagent 3 and 2 drops of hydrogen peroxide in 5 mL of dH2O. Peroxidase substrate should be freshly prepared immediately before use and cannot be stored.</li>
</ol>
2. Preparation of skin for wounding
NOTE: These steps should be performed in a class II laminar flow biosafety cabinet.
<ol>
	<li>Collect the skin in holding media and transport to the BSL-2 cabinet.</li>
	<li>Place the skin dermis side down within a 90 mm sterile Petri dish and remove adipose tissue with sterile scissors.</li>
	<li>Place the skin in a 50 mL tube containing 25 mL of HBSS with antibiotics for 10 min at RT. Shake intermittently to remove any residual blood and adipose tissue.</li>
	<li>Repeat step 2.3 using a new 50 mL tube.</li>
	<li>Place the skin in a fresh 50 mL tube containing 25 mL of HBSS, this time without antibiotics for 10 min at RT. Shake as in step 2.3.</li>
	<li>Perform a final skin rinse by placing skin in a new tube with 25 mL of DPBS. The skin is now ready to wound.</li>
</ol>
3. Creating ex vivo human skin wounds
NOTE: These steps should be performed in a class II laminar flow biosafety cabinet.
<ol>
	<li>Prepare the skin culture dishes prior to wounding. In a 60 mm Petri dish, stack two sterile absorbent pads and add 4 mL of human skin media via the side of the dish. Place a sterile nylon filter membrane onto the absorbent pad stack. 
	NOTE: Skin media can be altered depending on the required treatment conditions. Up to three wound explants may be cultured on each stack.</li>
	<li>Dry the dermal side of the skin on sterile gauze in a 90 mm Petri dish to remove residual DPBS. 
	NOTE: This prevents the skin from sliding around when wounding.</li>
	<li>Place the skin dermis side down on a clean 90 mm Petri dish lid and dab the epidermis dry with fresh sterile gauze. 
	NOTE: It is easier to wound the skin in a Petri dish lid than the base. Subsequent work should be carried out quickly to prevent the skin drying out.</li>
	<li>Holding the skin taut, press a 2 mm biopsy punch against the skin and twist gently. Do not punch entirely through the skin. 
	NOTE: Partial thickness wounds are designed to punch through the epidermis and partially into the dermis. There may be donor-to-donor and site-to-site variability in the force required to create the partial thickness wound.</li>
	<li>Use curved toothed tissue forceps to pick up each side of the 2 mm wound and hook curved iris scissors under the 2 mm wound to cut it out uniformly.</li>
	<li>Biopsy around the central 2 mm wound using a 6 mm biopsy punch to create a 6 mm explant with a partial thickness 2 mm wound in the center. 
	NOTE: A 6 mm biopsy punch may be used to score the skin to mark out where each 2 mm wound should be. Take care not to pierce through the tissue entirely. Create wound explants in a honeycomb pattern to reduce wastage.</li>
	<li>Place wound explants epidermis side up on the nylon filter membrane stack (prepared in step 3.1). 
	&#8203;NOTE: When handling wound explants, be careful not to damage the central wound. Use small forceps and pick up each explant at opposite sides.</li>
	<li>Incubate wounds at 32-37 &#176;C and 5% CO2 in a humidified atmosphere (90-95%) for 1-7 days. Replace the media every 2-3 days.</li>
</ol>
4. Whole-mount staining of ex vivo wounds
NOTE: This section describes immunofluorescence and immunoperoxidase staining methods. Mix all reagents well before use.
<ol>
	<li>Fluorescent staining method
	<ol>
		<li>Collect wound explants in 1.5 mL microcentrifuge tubes containing 500 &#181;L of skin fixative and incubate at 4 &#176;C overnight. 
		NOTE: The fixative used in this protocol works well for the described antibodies. Optimization will be required for other antibodies. Tissue fixation longer than 24 h may lead to over-fixation.</li>
		<li>The following day remove the fixative and replace with 1 mL of staining wash buffer. Biopsies can be stored in staining wash buffer at 4 &#176;C up to 2 weeks prior to staining. 
		&#8203;NOTE: For all wash buffer steps, use a serological pipette or pipette tip, taking care not to damage the wound.</li>
		<li>Aspirate the staining wash buffer and perform one more rinse with 1 mL of staining wash buffer.</li>
		<li>Calculate the amount of blocking buffer required for steps 4.1.5-4.1.6 (number of samples x 300 &#181;L = amount of blocking buffer in &#181;L). Make extra buffer if required.</li>
		<li>Add 150 &#181;L of blocking buffer to each sample and incubate for 1 h at RT. For all staining steps, ensure each sample is sufficiently covered and that there are no bubbles covering the biopsy wound surface. 
		&#8203;NOTE: This step onwards can be performed in 1.5 mL microcentrifuge tubes or in a 48 well plate. If using a 48 well plate, incubate the wounds face down in each well.</li>
		<li>Dilute the primary antibody in the remaining blocking buffer. 
		&#8203;NOTE: Anti-mouse keratin 14 (K14) diluted 1:1,000 in blocking buffer works well. Optimize this step for use with other antibodies or multiple probes.</li>
		<li>Aspirate the blocking buffer and add 150 &#181;L primary antibody per well/microcentrifuge tube. Incubate wound explants in primary antibody at 4 &#176;C overnight.</li>
		<li>The next day, aspirate the primary antibody and rinse in staining wash buffer containing 0.2% sodium azide for 1 h at RT (500 &#181;L per sample).</li>
		<li>Perform three more rinsing steps using staining wash buffer (30 min per wash, 500 &#181;L per sample).</li>
		<li>Dilute the fluorescently conjugated secondary antibody in staining wash buffer (e.g., goat anti-mouse 488 at 1:400 dilution).</li>
		<li>Calculate the required amount of secondary antibody (number of samples x 150 &#181;L = amount in &#181;L).</li>
		<li>Add 150 &#181;L of secondary antibody to each well/microcentrifuge tube. Incubate for 1 h at RT. Perform incubation steps 4.1.10 - 4.1.16 in the dark as the secondary antibody is light sensitive. 
		&#8203;NOTE: This step can be performed at 4 &#176;C overnight if required. Optimize the concentration of secondary antibody required for adequate signal and limited background staining.</li>
		<li>Remove the secondary antibody and perform 3 x 30 min rinses with staining wash buffer (500 &#181;L per sample).</li>
		<li>Discard the leftover wash buffer and calculate the amount of DAPI working solution required (as per step 4.1.11).</li>
		<li>Counterstain each explant with 150 &#181;L of DAPI working solution for 10 min at RT. 
		&#8203;NOTE: DAPI will stain cell nuclei blue. Hoechst dye can be used as an alternative to DAPI.</li>
		<li>Perform two final 30 min washes with staining wash buffer (500 &#181;L per sample). Biopsies can be stored in staining wash buffer at 4 &#176;C in the dark up to two weeks prior to imaging.</li>
	</ol>
	</li>
	<li>Brightfield staining method.
	<ol>
		<li>Perform steps 4.1.1 - 4.1.3.</li>
		<li>Quench endogenous peroxidase activity with peroxidase block at 4 &#176;C overnight. 
		NOTE: This step is important when using an HRP-conjugated antibody to reduce non-specific background staining from the tissue. Highly vascularized tissue will contain more endogenous peroxidase activity.</li>
		<li>Discard the peroxidase block and rinse twice for 30 min in staining wash buffer.</li>
		<li>Perform steps 4.1.4 - 4.1.8. 
		&#8203;NOTE: Washes after step 4.1.7 are particularly important to remove sodium azide from the samples. If sodium azide is not adequately removed, it will inactivate the HRP and interfere with staining detection.</li>
		<li>Add 150 &#181;L HRP-conjugated secondary antibody to each well/microcentrifuge tube and incubate overnight at 4 &#176;C or 1 h at RT.</li>
		<li>Remove the secondary antibody and perform 3 x 30 min washes in staining wash buffer.</li>
		<li>Add 150 &#181;L ABC reagent to each well/microcentrifuge tube and incubate overnight at 4 &#176;C or 1 hour at RT.</li>
		<li>Aspirate the ABC reagent and perform 3 x 30 min washes in staining wash buffer.</li>
		<li>Add 150 &#181;L peroxidase substrate to one explant and determine the time required to detect a noticeable color change. 
		&#8203;NOTE: Choose a sample where strong staining is expected. In this case, a red ring to show the migrating epidermis (K14). 3,3&#39;-diaminobenzidine-4, or any other appropriate chromogenic substrate, may be used as a replacement for this peroxidase substrate.</li>
		<li>Once a color change is observed, remove the peroxidase substrate, and replace with 1 mL of dH2O.</li>
		<li>Repeat the peroxidase substrate detection for the other explants, incubating for the time determined in step 4.2.11.</li>
		<li>Rinse all explants with 1 mL of dH2O to remove residual peroxidase substrate. Although explants may be stored up to one week at 4 &#176;C prior to imaging, it is better to image them as soon as possible to prevent leaching of the peroxidase substrate into the dH2O over time.</li>
	</ol>
	</li>
</ol>
5. Imaging and quantification
<ol>
	<li>Fluorescent imaging 
	NOTE: Fluorescent imaging is performed using a confocal laser-scanning microscope. However, an inverted fluorescent microscope may be sufficient for acquiring 2D images to quantify wound closure rates. When selecting secondary antibodies, ensure that the chosen fluorochromes are compatible with the excitation and emission spectra of the microscopy equipment available.
	<ol>
		<li>Use a confocal laser-scanning microscope equipped with a 2.5x, 10x and 20x objective, x-y-z motorized stage, digital camera, and acquisition software. Switch on the transmitted light detector (TPMT) to allow easy visualization of each biopsy and to enable measurement of total wound closure. Alternatively, measure each wound via brightfield microscopy following fluorescence imaging.</li>
		<li>Place a 60 mm Petri dish base on the imaging platform and add a thin layer (around 1 mL) of DPBS. 
		&#8203;NOTE: If too much DPBS is used, the biopsy will move around during imaging. Alternatively use a 48 well plate if a plate holder is available.</li>
		<li>Use small tissue forceps to transfer wound explants from wells/microcentrifuge tubes to the Petri dish containing DPBS. Place the biopsy wound side down in the Petri dish.</li>
		<li>Use the eyepiece and fluorescent lamp to locate and focus on the wound. If bubbles are trapped under the sample in the field of view, pick up the wound with tissue forceps and reposition.</li>
		<li>Set up the imaging software, ensuring equal pinhole size between channels for optimum confocality. For this, check the value of one airy unit for each channel and select the largest value. Select scan speed, image quality and averaging. 
		&#8203;NOTE: The fluorochromes of the conjugated secondary antibodies and the chosen counterstain (e.g., DAPI) will dictate the channels required.</li>
		<li>Switch on the live acquisition software and adjust the laser power and gain of each channel to the levels required to visualize staining. Reduce background noise by increasing the digital offset.</li>
		<li>Position the wound in the center of the imaging plane. 
		&#8203;NOTE: If the wound does not fill the entire image due to using a smaller objective or creating a larger wound, take a panel of images and stitch them together (manually or with a tiling function in the relevant imaging software).</li>
		<li>Acquire images of the wound biopsies. Use the same imaging settings between explants. 
		NOTE: Higher power images will allow assessment of tissue structures and cellular marker expression and location.</li>
		<li>Collect serial Z stacks through the wound, especially where the tissue is not completely flat against the Petri dish. Use analysis software to collapse the Z stack into a single maximum intensity projection image.</li>
	</ol>
	</li>
	<li>Brightfield imaging 
	NOTE: Brightfield imaging of immunoperoxidase stained biopsies can be performed in multiple ways.
	<ol>
		<li>Inverted microscope imaging: Prepare wound explants for imaging by placing them in a Petri dish/well as described in steps 5.1.2-5.1.3. Acquire digital images under brightfield illumination on an inverted microscope equipped with a digital camera. Stitch together multiple images if required.</li>
		<li>Wireless digital microscope imaging: Use a wireless digital microscope connected to a phone or laptop to obtain high quality images in a cost-effective manner. Place explants wound side up onto some tissue and remove any residual dH2O (or staining wash buffer) from sample storage. Position the wound explant in the center of the microscope field of view. Acquire images using the connected camera.</li>
	</ol>
	</li>
	<li>Quantification 
	&#8203;NOTE: Percentage wound closure can be quantified in any software that allows freehand shapes to be drawn and measured. ImageJ can be used to perform quantification as follows:
	<ol>
		<li>Open the image to be quantified in ImageJ software.</li>
		<li>Use the freehand shape tool to draw around the outside of the re-epithelialized wound where it meets the normal skin. Press M (or Analyze | Measure) to acquire an &#39;outer&#39; area measurement. 
		NOTE: The re-epithelialized wound tissue texture differs from normal skin. The images do not need to be scaled prior to this type of analysis.</li>
		<li>Use the freehand shape tool to draw around the open wound area. This is where the open wound meets the inside edge of the re-epithelializing tissue. Press M (or Analyze | Measure) to acquire an &#39;inner&#39; area measurement.</li>
		<li>Use the following equation to deduce percentage wound re-epithelialization/closure: 
		% Closure = (Outer Wound Area - Inner Wound Area) / (Outer Wound Area) x 100 
		NOTE: Percentage area coverage of antibodies can be deduced in the same way (e.g., K14) or as a percentage of the total wound area. Percentage intensity can also provide semi-quantitative information about tissue level expression of markers of interest, while high power imaging presents expression data at the cellular level.</li>
	</ol>
	</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+isolation+from+human+skin+for+phenotypic+analysis+and+ex+vivo+cell+culture.">Lymphocyte isolation from human skin for phenotypic analysis and ex vivo cell culture.</a> Journal of Visualized Experiments. (110), e52564 (2016).</li><li>Pupovac, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+immunocompetent+3D+skin+models.">Toward immunocompetent 3D skin models.</a> Advanced Healthcare Materials. 7 (12), 1701405 (2018).</li><li>Ata&#231;, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+and+hair+on-a-chip:+in+vitro+skin+models+versus+ex+vivo+tissue+maintenance+with+dynamic+perfusion.">Skin and hair on-a-chip: in vitro skin models versus ex vivo tissue maintenance with dynamic perfusion.</a> Lab on a Chip. 13 (18), 3555-3561 (2013).</li><li>Kim, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microscale,+full-thickness,+human+skin+on+a+chip+assay+simulating+neutrophil+responses+to+skin+infection+and+antibiotic+treatments.">A microscale, full-thickness, human skin on a chip assay simulating neutrophil responses to skin infection and antibiotic treatments.</a> Lab on a Chip. 19 (18), 3094-3103 (2019).</li><li>Jardet, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+human+Th17-driven+ex+vivo+skin+inflammation+model.">Development and characterization of a human Th17-driven ex vivo skin inflammation model.</a> Experimental Dermatology. 29 (10), 993-1003 (2020).</li><li>Chen, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metformin+attenuates+diabetes-induced+tau+hyperphosphorylation+in+vitro+and+in+vivo+by+enhancing+autophagic+clearance.">Metformin attenuates diabetes-induced tau hyperphosphorylation in vitro and in vivo by enhancing autophagic clearance.</a> Experimental Neurology. 311, 44-56 (2019).</li><li>Demirovic, D., Rattan, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curcumin+induces+stress+response+and+hormetically+modulates+wound+healing+ability+of+human+skin+fibroblasts+undergoing+ageing+in+vitro.">Curcumin induces stress response and hormetically modulates wound healing ability of human skin fibroblasts undergoing ageing in vitro.</a> Biogerontology. 12 (5), 437-444 (2011).</li><li>Wilkinson, H. N., Hardman, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+senescence:+A+functional+link+between+diabetes+and+ageing.">Wound senescence: A functional link between diabetes and ageing.</a> Experimental Dermatology. 30 (1), 68-73 (2020).</li><li>Fisher, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+fragmentation+promotes+oxidative+stress+and+elevates+matrix+metalloproteinase-1+in+fibroblasts+in+aged+human+skin.">Collagen fragmentation promotes oxidative stress and elevates matrix metalloproteinase-1 in fibroblasts in aged human skin.</a> The American Journal of Pathology. 174 (1), 101-114 (2009).</li><li>Quan, T., Little, E., Quan, H., Voorhees, J. J., Fisher, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+matrix+metalloproteinases+and+collagen+fragmentation+in+photodamaged+human+skin:+impact+of+altered+extracellular+matrix+microenvironment+on+dermal+fibroblast+function.">Elevated matrix metalloproteinases and collagen fragmentation in photodamaged human skin: impact of altered extracellular matrix microenvironment on dermal fibroblast function.</a> Journal of Investigative Dermatology. 133 (5), 1362 (2013).</li></ol>

The authors declare no conflicts of interest.

In this experimental protocol, we describe an optimized method for evaluating wound closure in human ex vivo skin using whole-mount tissue staining. This is an important resource to allow critical evaluation of potential wound treatments, and to provide better understanding of the human wound repair response. We have published healing assessment in ex vivo skin wounds previously12,13, yet in these reports the whole-mount staining approach was not used to measure wound closure. Whole-mount staining is far easier and requires less technical experience than standard histology, which involves paraffin or OCT embedding and sectioning of samples. The whole-mount procedure also reduces experimental variability, allowing quantification of the entire wound and not just a single transverse section at a defined position within the tissue (see Figure 4B for comparative illustration). We fully support the importance of quantifying healing of the entire non-symmetrical wound structure, as clearly outlined by Rhea and Dunnwald for murine acute wounds14. These authors showed the importance of serially sectioning in vivo excisional wounds for reproducible and precise measurements of wound morphology. Serial sectioning could equally be applied to human ex vivo wounds; however, for accurate quantification of wound closure and re-epithelialization, high throughput whole-mount staining should be the preferred method. We note that this whole-mount staining protocol should also be compatible with subsequent processing (wax or OCT) for traditional histological analysis.
Whole-mount staining is not without disadvantages. While it affords higher reproducibility in wound healing experiments, it does require the use of more tissue for analysis than standard histological techniques. This may be an issue where tissue access is limited, particularly where multiple antibodies need to be assessed. An alternative approach would be to employ an incisional wounding method where wound width is relatively uniform and variability is reduced (as shown in mouse and human wounds15,16). However, excisional wounds remain more applicable to most pathological wound types17.
In this study, 2 mm partial thickness wounds were created within the center of 6 mm skin explants. This method may be optimized for alternative excisional wound and explant sizes at different skin depths18. In addition, the force required to generate wounds will vary between donors, where aged skin will require less force to biopsy. We would also avoid using skin displaying prominent stretch marks or other structural alterations. We have validated a range of antibodies to consider different aspects of the ex vivo healing response. This protocol may also be used with other skin-relevant antibodies, where antibody concentrations and incubation times will need to be optimized. Nevertheless, we believe our protocol is most suited to absolute quantification of total wound closure, followed by spatial assessment of specific proteins of interest. While whole-mount provides reduced resolution of immunolocalization versus standard histological analysis of tissue sections, it provides additional 3D information that is missing from standard 2D histology.
One caveat of assessing healing in ex vivo skin versus in vivo models is that it lacks a systemic response. An important aspect of wound repair is inflammation and subsequent tissue granulation, which is caused by an influx of inflammatory cells and endothelial cells from the vasculature19. Despite this limitation, ex vivo skin still provides a better recapitulation of clinical healing than cell-based wound assays. In vitro experiments in general involve single cell type monolayers or co-cultures grown on tissue culture plastic, whereas ex vivo skin provides a native environment to explore cell behavior. More recently, a number of skin equivalent systems have emerged, where skin is grown in a laboratory setting from artificial matrix and isolated skin cells20,21. Although these models mimic human skin better than most in vitro approaches, they still do not fully simulate the native tissue environment and are generally too fragile to injure reproducibly. Additionally, we (and others) have demonstrated that ex vivo human skin tissue retains resident immune cells, which will no doubt contribute to repair22,23. Future work should now focus on extending the viability and immunocompetency of the ex vivo model for late-stage healing assessment24. One option is further advancement of promising organ-on-a-chip technologies capable of prolonging tissue viability and maintaining native skin architecture for up to two weeks in culture25. Ex vivo models have also begun to consider the importance of the skin inflammatory response by successfully incorporating immune cells, such as neutrophils, into the host tissue26 or injecting host tissue with antibodies to elicit an immune reaction27. We expect that these findings will pave the way for development of more refined and translatable methods in the future.
A major benefit of using ex vivo skin to measure wound closure is the ability to compare healing rates in healthy (e.g., non-diabetic) versus pathological (e.g., diabetic or aged) tissue. Here we showed that re-epithelialization and barrier formation are indeed impaired in diabetic versus healthy ex vivo&#160;wounds. Indeed, this provides a route for pre-clinical assessment of pathological repair, where ageing and diabetes are major risk factors for developing chronic wounds1. While in vitro pathological models exist, such as cells isolated from aged and diabetic tissue, or cells cultured in high glucose to mimic hyperglycemia28,29, these cells can quickly lose their phenotype once removed from the in vivo microenvironment. An important component of the extrinsic pathological healing environment is the dermal matrix, which is altered in both ageing and diabetes30. Indeed, this perturbed matrix affects the behavior of resident and na&#239;ve fibroblasts31,32. Thus, the importance of studying cells in their host tissue environment cannot be underestimated.
In summary, our protocol provides an important platform to quantify human wound re-epithelialization, explore regulatory factors and to test the validity and efficacy of potential therapeutics12,13. While pre-clinical testing does still require in vivo approaches, a combined strategy using ex vivo human tissue and in vivo murine wounding should refine the pre-clinical pathway, reducing animal use while increasing cross-species translatability.

Chronic, non-healing wounds, which are highly prevalent in the elderly and diabetic, are a majorly unappreciated area of clinical unmet need. These wounds present a major physical and psychological burden to patients and cost healthcare providers billions each year to treat1. Despite improved understanding of wound biology and advancements in technology, up to 40% of chronic wounds still fail to heal following best standard care2. Thus, 14-26% of patients with diabetic foot ulcers subsequently require amputation3, while 5-year post-amputation mortality rate stands at approximately 70%4. As a result, there is an urgent requirement to develop efficacious new therapies to improve patient quality of life while reducing the substantial healthcare burden imposed by poor healing wounds. Poorly predictive pre-clinical models remain a significant hurdle to the development of effective new therapies.
Wound repair is a dynamic and multifaceted process involving a diverse range of cell types, countless levels of communication and a tissue environment that is temporally remodeled. Skin healing is underpinned by four major reparative stages: hemostasis, inflammation, proliferation, and matrix remodeling. These stages ultimately act to prevent blood loss and infection, close the wound surface (a process termed re-epithelialization) and return the skin to an uninjured state5. Chronic wounds are associated with diverse etiology and widespread perturbation to healing processes6, further complicating the identification of therapeutic targets. Nevertheless, a broad range of models have been developed to both elucidate the molecular and cellular drivers of wound pathology and test new therapeutic approaches7.
The most used wound repair model is acute wounding in the mouse. Mice are highly tractable for mechanistic studies and provide validated models of ageing and diabetes8. Despite the general similarities shown amongst mouse and human healing, between-species differences in skin structure and healing dynamics remain. This means most murine wound research does not easily translate to the clinic9. Consequently, there has been a push towards human in vitro and ex vivo systems with high applicability and translatability10,11.
Here we provide an in-depth protocol for performing partial thickness excisional wounds in ex vivo human skin. We&#160;also&#160;outline our whole-mount staining approach as a highly reproducible method of evaluating ex vivo human skin healing. We show the trajectory of epidermal repair (re-epithelialization) and subsequent barrier formation, evaluating the rate of wound closure in healthy versus diabetic human skin. Finally, we demonstrate how whole-mount staining can be adapted for use with a range of antibodies to assess various aspects of the healing response.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>50 mL Falcon Tubes</td>
			<td>Falcon</td>
			<td>352070</td>
			<td>For skin washing</td>
		</tr>
		<tr>
			<td>1.5&#160;ml TubeOne Microcentrifuge Tubes, Natural (Sterile)</td>
			<td>Starlab</td>
			<td>S1615-5510</td>
			<td>For whole-mount staining</td>
		</tr>
		<tr>
			<td>48-Well CytoOne Plate, TC-Treated</td>
			<td>Starlab</td>
			<td>CC7682-7548</td>
			<td>For whole-mount staining</td>
		</tr>
		<tr>
			<td>Acetic Acid Glacial</td>
			<td>Fisher Chemical</td>
			<td>A/0400/PB15</td>
			<td>Part of fixative</td>
		</tr>
		<tr>
			<td>Alkyltrimethylammonium Bromide</td>
			<td>Sigma-Aldrich</td>
			<td>M7635</td>
			<td>Part of fixative</td>
		</tr>
		<tr>
			<td>Anti-Alpha Smooth Muscle Actin Antibody [1A4]</td>
			<td>Abcam</td>
			<td>ab7817</td>
			<td>Stains blood vessels</td>
		</tr>
		<tr>
			<td>Anti-Collagen I Antibody</td>
			<td>Abcam</td>
			<td>ab34710</td>
			<td>Stains collagen</td>
		</tr>
		<tr>
			<td>Anti-Cytokeratin 14 Antibody [LL002]</td>
			<td>Abcam</td>
			<td>ab7800</td>
			<td>Stains epidermis</td>
		</tr>
		<tr>
			<td>CD1A Antibody (CTB6)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-5265</td>
			<td>Stains Langerhans cells</td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-diamidino-2-phenylindole, dihydrochloride)</td>
			<td>Thermo Fisher Scientific</td>
			<td>62247</td>
			<td>Counterstain for cell nuclei</td>
		</tr>
		<tr>
			<td>Falcon 60mm Petri dishes</td>
			<td>Falcon</td>
			<td>353004</td>
			<td>Human ex vivo culture</td>
		</tr>
		<tr>
			<td>Fibronectin Antibody (EP5)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-8422</td>
			<td>Stains fibronectin</td>
		</tr>
		<tr>
			<td>Formaldehyde, Extra Pure, Solution 37-41%, SLR</td>
			<td>Fisher Chemical</td>
			<td>F/1501/PB17</td>
			<td>Part of fixative</td>
		</tr>
		<tr>
			<td>Gauze Swabs</td>
			<td>Medisave</td>
			<td>CS1650</td>
			<td>To clean skin</td>
		</tr>
		<tr>
			<td>Gibco&#8482; Antibiotic-Antimycotic Solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>15240062</td>
			<td>Human ex vivo culture</td>
		</tr>
		<tr>
			<td>Gibco DMEM, high glucose, no glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>11960044</td>
			<td>Human ex vivo culture</td>
		</tr>
		<tr>
			<td>Gibco Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>10500064</td>
			<td>Human ex vivo culture</td>
		</tr>
		<tr>
			<td>Gibco HBSS, no calcium, no magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>14170088</td>
			<td>Human ex vivo culture</td>
		</tr>
		<tr>
			<td>Gibco L-Glutamine (200 mM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030081</td>
			<td>Human ex vivo culture</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide</td>
			<td>Sigma-Aldrich</td>
			<td>H1009-100ML</td>
			<td>For immunoperoxidase staining</td>
		</tr>
		<tr>
			<td>ImageJ Software</td>
			<td>National Institutes of Health</td>
			<td>N/A</td>
			<td>For image analysis</td>
		</tr>
		<tr>
			<td>Invitrogen IgG (H+L) Cross-Adsorbed Goat anti-Mouse, Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11001</td>
			<td>Secondary antibody used depends on required fluorochromes and primary antibody</td>
		</tr>
		<tr>
			<td>Invitrogen IgG (H+L) Cross-Adsorbed Goat anti-Rabbit, Alexa Fluor 594</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11012</td>
			<td>Secondary antibody used depends on required fluorochromes and primary antibody</td>
		</tr>
		<tr>
			<td>Invitrogen LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>L3224</td>
			<td>For viability assessment of tissue</td>
		</tr>
		<tr>
			<td>Iris Forceps, 10 cm, Curved, 1x2 teeth</td>
			<td>World Precision Instruments</td>
			<td>15917</td>
			<td>To create wounds</td>
		</tr>
		<tr>
			<td>Iris Scissors, 11 cm, Curved, SuperCut, Tungsten Carbide</td>
			<td>World Precision Instruments</td>
			<td>501264</td>
			<td>To create wounds</td>
		</tr>
		<tr>
			<td>Iris Scissors, 11 cm, Straight, SuperCut, Tungsten Carbide</td>
			<td>World Precision Instruments</td>
			<td>501263</td>
			<td>To remove adipose tissue</td>
		</tr>
		<tr>
			<td>Keratin 1 Polyclonal Antibody, Purified</td>
			<td>Biolegend</td>
			<td>905201</td>
			<td>Stains epidermis</td>
		</tr>
		<tr>
			<td>Keratin 14 Polyclonal Antibody, Purified</td>
			<td>Biolegend</td>
			<td>905301</td>
			<td>Stains epidermis</td>
		</tr>
		<tr>
			<td>LSM 710 Confocal Laser Scanning Microscope</td>
			<td>Carl Zeiss</td>
			<td>Discontinued</td>
			<td>For fluorescent imaging</td>
		</tr>
		<tr>
			<td>Merck Millipore Absorbent pads</td>
			<td>Merck Millipore</td>
			<td>AP10045S0</td>
			<td>Human ex vivo culture</td>
		</tr>
		<tr>
			<td>Merck Millipore Nylon Hydrophilic Membrane Filters</td>
			<td>Merck Millipore</td>
			<td>HNWP04700</td>
			<td>Human ex vivo culture</td>
		</tr>
		<tr>
			<td>Normal Goat Serum Solution</td>
			<td>Vector Laboratories</td>
			<td>S-1000-20</td>
			<td>Animal serum used depends on secondary antibody</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Solution</td>
			<td>Sigma-Aldrich</td>
			<td>P3619</td>
			<td>For wash buffer</td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td>For blocking buffer</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Bioreagents</td>
			<td>BP358-212</td>
			<td>Part of fixative</td>
		</tr>
		<tr>
			<td>Sterilisation Pouches</td>
			<td>Medisave</td>
			<td>SH3710</td>
			<td>To sterilise instruments</td>
		</tr>
		<tr>
			<td>Stiefel 2mm biopsy punches</td>
			<td>Medisave</td>
			<td>BI0500</td>
			<td>For partial thickness wound</td>
		</tr>
		<tr>
			<td>Stiefel 6mm biopsy punches</td>
			<td>Medisave</td>
			<td>BI2000</td>
			<td>For outer explant</td>
		</tr>
		<tr>
			<td>Thermo Scientific Sterilin Standard 90mm Petri Dishes</td>
			<td>Thermo Fisher Scientific</td>
			<td>101VR20</td>
			<td>To prepare skin</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Chemical</td>
			<td>T/3751/08</td>
			<td>For wash buffer</td>
		</tr>
		<tr>
			<td>VECTASTAIN Elite ABC-HRP Kit, Peroxidase (Rabbit IgG)</td>
			<td>Vector Laboratories</td>
			<td>PK-6101</td>
			<td>For immunoperoxidase staining; HRP kit used depends on primary antibody</td>
		</tr>
		<tr>
			<td>Vector NovaRED Substrate Kit, Peroxidase (HRP)</td>
			<td>Vector Laboratories</td>
			<td>SK-4800</td>
			<td>For immunoperoxidase staining</td>
		</tr>
		<tr>
			<td>Wireless Digital Microscope</td>
			<td>Jiusion</td>
			<td>N/A</td>
			<td>For brightfield imaging</td>
		</tr>
	</tbody>
</table>

human ex vivo wound model and whole-mount staining approach to accurately evaluate skin repair

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic wound treatments are inadequate, while available pre-clinical models poorly predict the clinical efficacy of new therapies. Here we describe a high throughput, pre-clinical model to assess multiple aspects of the human skin repair response. Partial thickness wounds were created in human ex vivo skin and cultured across a healing time course. Skin wound biopsies were collected in fixative for the whole-mount staining procedure. Fixed samples were blocked and incubated in primary antibody, with detection achieved via fluorescently conjugated secondary antibody. Wounds were counterstained and imaged via confocal microscopy before calculating percentage wound closure (re-epithelialization) in each biopsy. Applying this protocol, we reveal that 2 mm excisional wounds created in healthy donor skin are fully re-epithelialized by day 4-5 post-wounding. On the contrary, closure rates of diabetic skin wounds are significantly reduced, accompanied by perturbed barrier reformation. Combining human skin wounding with a novel whole-mount staining approach allows a rapid and reproducible method to quantify ex vivo wound repair. Collectively, this protocol provides a valuable human platform to evaluate the effectiveness of potential wound therapies, transforming pre-clinical testing and validation.

In this report, we present a novel ex vivo skin wounding and whole-mount staining approach to assess factors that influence the human skin repair response. Figure 1A shows a schematic of the procedural pipeline, which can be performed in 3-10 days, depending on wound incubation times. The partial thickness wounds are cultured on membrane stacks at the air : membrane interface and can be collected for whole-mount staining, embedded in paraffin or OCT medium for general histology, or frozen in liquid nitrogen for biochemical analysis (Figure 1B). We generally create 2 mm partial thickness wounds within the center of 6 mm explants. However, the size of the wound and surrounding explant may be altered depending on requirements. The whole-mount procedure has been successfully adapted for both immunoperoxidase and immunofluorescence staining methods (Figure 1C).
Immunofluorescence allows for the probing of tissue with multiple antibodies. For this, we advise using primary antibodies raised in different species, and species-matched fluorescently conjugated secondary antibodies to limit cross-species reactivity. Antibody concentrations and incubation times will need to be optimized. If background staining is observed, reduce antibody concentrations, increase wash steps, and add blocking buffer to the secondary antibody. Fresh tissue viability can be directly assessed with commercial viability dyes (see Table of Materials). We also show that tissue may be fixed post viability staining and successfully imaged when it is practically suitable (Figure 1D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62326/62326fig01.jpg" /> 
Figure 1: The human ex vivo wounding and whole-mount staining approach. (A) Pipeline depicting the procedural workflow from collecting skin and performing ex vivo wounding, to staining tissue and analyzing data. (B) Diagram demonstrating the human ex vivo skin wound culture system with analyses routinely performed on the tissue. (C) Whole-mount staining can be employed using both immunoperoxidase and immunofluorescence techniques. K14 = keratin 14. (D) Live tissue may be stained with commercial viability dyes and imaged successfully post fixation. Bar = 100 &#181;m. This staining was performed in non-diabetic skin. <a href="https://www.jove.com/files/ftp_upload/62326/62326fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The most widely applicable use for whole-mount staining of wounds is to determine wound closure rate in a more reproducible manner than can be provided via histological sectioning. Percentage closure was quantified as percentage re-epithelialization of the wound surface, as demonstrated in Figure 2A. Percentage area coverage of specific markers can be measured from the total wound area or as a percentage of the re-epithelialized wound. We characterized healing in healthy (non-diabetic) versus diabetic skin across a time course of seven days, collecting wounds at each day post-wounding (representative images, Figure 2B). Healthy skin wounds closed over time as expected, with full closure observed in most samples by day 4-5. On the contrary, diabetic skin wounds failed to close fully within the seven-day analysis period (Figure 2C). A significant delay in wound closure was observed between healthy and diabetic skin wounds when comparing healing rates at each time-point post-injury (P &#60; 0.001 to day 6, P &#60; 0.05 at day 6 and P &#60; 0.05 to P &#60; 0.001 at day 7).
Following assessment of overall wound closure rates; we measured the percentage of the entire wound area (outer area in Figure 2A) where K14 positive cells could be visualized (green staining in Figure 2B). Interestingly, we observed that in healthy ex vivo skin wounds, K14 staining peaked at day 2 and then rapidly declined (significance at each time-point versus the day 2 peak, Figure 2D). This is likely reflecting re-formation of the early epidermal barrier, excluding K14 antibody penetration through differentiated epidermal layers (see Figure 2E schematic). During the re-epithelialization process, basal layer (K14+ve) keratinocytes migrate inwards over the open wound such that the epidermis closer to the outer wound edge forms earlier than the epidermis closer to the inner wound edge (migrating front). While the front edge of the newly formed epidermis continues to migrate to close the remaining open wound, the outer edge epidermis begins to differentiate to reform the other epidermal layers. In early healing, we would thus expect to see most of the re-epithelialized area consists of basal (K14+ve) cells, while in later repair K14 staining is lost as the epidermis differentiates from the outside inwards (see whole-mount images in Figure&#160;2E). Therefore, the decline in K14 staining shown in Figure 2D (downward arrows) correlates with increased epidermal differentiation. Interestingly, visible K14 staining peaked earlier in healthy (day 2) versus diabetic (day 4) wounds, further demonstrating that re-epithelialization and subsequent epidermal differentiation are delayed in diabetic skin wounds.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62326/62326fig02.jpg" /> 
Figure 2: Whole-mount staining reveals perturbed healing rates in diabetic versus healthy skin. (A) The method used to quantify wound closure from outer and inner wound measurements. Brightfield images show keratin 14 (K14) staining in red. Bar = 300 &#181;m. (B) Representative images of healing over time (day post-wounding) in healthy and diabetic skin. Bar = 500 &#181;m. K14 = green. DAPI = blue nuclei. (C) Quantification of wound closure rates (percentage re-epithelialization) showing that ex vivo wounds from healthy skin close significantly faster than ex vivo&#160;wounds from diabetic skin. H = healthy. Db = diabetic. (D) Percentage K14 staining peaks earlier in healthy versus diabetic skin and then declines in line with increased epidermal differentiation (down arrows). (E) K14 (basal epidermal cell) staining is lost as the epidermis differentiates. D = differentiated. ND = not differentiated. White dotted lines depict inner and outer wound edges. White arrows = direction of migration. n = 6 wounds per donor, per time point. Mean +/- SEM. * = P &#60; 0.05, ** = P &#60; 0.01 and *** = P &#60; 0.001. Healthy and diabetic compared at each healing time point in C (P value for least significant comparison). Temporal change in K14 staining compared to peak for each donor in D. <a href="https://www.jove.com/files/ftp_upload/62326/62326fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
We next used whole-mount staining to explore tissue expression and localization of other wound-relevant markers in non-diabetic skin (Figure&#160;3). All antibodies used and their working concentrations are provided in the Table of Materials. Blood vessels in the open wound stained positively with alpha smooth muscle actin (a-SMA) antibody, used in combination with K14 to delineate the epidermal edges in lower power images (Figure 3A). The dermal matrix was stained with antibodies against collagen type I (COL 1) and fibronectin (Fn). Here collagen was observed as abundant thick fibers while fibronectin fibers were sparse, wavy, and thin (Figure 3A). Our whole-mount staining approach is also able to provide cell level resolution of staining, as demonstrated for K14-positive keratinocytes (Figure 3B).
Finally, we show that human ex vivo wounds possess resident immune cells, with Langerhans cells detected around newly formed epidermis at day 3 post-wounding (Figure 3C). Indeed, these results suggest that whole-mount staining may be used to investigate key features of the healing response including inflammation, proliferation, and the extracellular matrix (Figure 4A). Taken together, our data reveal that the combined ex vivo skin wounding and whole-mount staining procedure is a valid method to assess various aspects of healthy and diabetic (pathological) human skin repair.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62326/62326fig03.jpg" /> 
Figure 3: Optimization of the whole-mount staining approach for use with other antibodies. (A) Blood vessels were stained with alpha smooth muscle actin (&#945;-SMA, green) and keratin 14 (K14, red), while matrix fibers were stained with collagen I (COL 1, red) and fibronectin (Fn, green). (B) The whole-mount procedure provides up to cell level resolution of localization (K14, green; K1, red). (C) CD1a+ve Langerhans cells (green) observed in newly formed epidermis. DAPI = blue nuclei. Bar = 100 &#181;m. White dotted lines show inner and outer wound edges and separate wound from epidermis. This staining was performed in non-diabetic skin. <a href="https://www.jove.com/files/ftp_upload/62326/62326fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62326/62326fig04.jpg" /> 
Figure 4: Validity of the whole-mount staining procedure for assessing wound healing. (A) Illustration depicting how the whole-mount staining technique can evaluate wound-relevant processes. Antibodies used = red text. K14 = keratin 14. COL 1 = collagen 1. Fn = fibronectin. (B) The whole-mount staining procedure (blue arrows) introduces less variability to wound closure measurements than standard histological analysis (red arrows). S1 = section 1. WE = wound edge. Bar = 300 &#181;m. This staining was performed in non-diabetic skin.&#160;<a href="https://www.jove.com/files/ftp_upload/62326/62326fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair at JoVE.com

Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair

Here we demonstrate an optimized technique for assessing wound repair using ex vivo human skin combined with a whole-mount staining approach. This methodology provides a pre-clinical platform for the evaluation of potential wound therapies.

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic ...

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6.7K Views.  University of Hull. Here we demonstrate an optimized technique for assessing wound repair using ex vivo human skin combined with a whole-mount staining approach. This methodology provides a pre-clinical platform for the evaluation of potential wound therapies.

Video: Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

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 ...

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an integral part of the male reproductive tract, is responsible for sperm storage, maturation, and transport. The adult epididymis is a highly coiled tube that develops from a simple and straight embryonic precursor known as Wolffian duct (WD). Proper coiling of the epididymis is essential for male fertility, as sperm in the testis are unable to fertilize an oocyte. However, the mechanism responsible for epididymal development and coiling remains unclear, partially due to the lack of whole organ culture and imaging methods. In this study, we describe an in vitro culture system and whole mount immunofluorescence protocol to better visualize the process of WD coiling and development, which may also be applied to study other tubular organs.

We present a method for isolation and culture of the mouse Wolffian duct (WD). We also demonstrate a detailed procedure for whole mount immunostaining of cultured/freshly isolated WDs with fluorescently tagged antibodies. Together, these techniques enable the study of WD development, coiling, and differentiation.

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, especially, in plant reproductive biology. However, plant morphological, anatomical, and ultrastructural analyses traditionally involve time-consuming embedding and sectioning procedures for bright field, scanning, and electron microscopy. Recent progress in confocal fluorescence microscopy, state-of-the-art 3-D computer-aided microscopic analyses, and the continuous refinement of molecular techniques to be used on minimally processed whole-mount specimens, has led to an increased demand for developing efficient and minimal sample processing techniques. In this protocol, we describe techniques for properly dissecting Arabidopsis flowers and siliques, basic clearing techniques, and some staining procedures for whole-mount observations of reproductive structures.

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

In this protocol, we describe techniques for the proper dissection of Arabidopsis flowers and siliques, some basic clearing techniques, and selected staining procedures for whole-mount observations of reproductive structures.

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch&#39;s membrane pathology is part of this process. Alterations in human Bruch&#39;s membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch&#39;s membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch&#39;s membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch&#39;s membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch&#039;s Membrane

The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch&#39;s membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix.

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...

Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching morphogenesis and patterning, as well as immune cell distribution at a cellular level. The analysis of peripheral nerve and blood vessel anatomical structures in adult tissues provides some insights into the understanding of functional neuro-vascular wiring and neuro-vascular degeneration in pathological conditions such as wound healing. As a highly informative model system, we have focused our studies on adult ear skin, which is readily accessible for dissection. Our simple and reproducible protocol provides an accurate depiction of the cellular components in the entire skin, such as peripheral nerves (sensory axons, sympathetic axons, and Schwann cells), blood vessels (endothelial cells and vascular smooth muscle cells), and inflammatory cells. We believe this protocol will pave the way to investigate morphological abnormalities in peripheral nerves and blood vessels as well as the inflammation in the adult ear skin under different pathological conditions.

Here, we describe a high resolution whole-mount imaging method in the entire adult mouse ear skin, which enables us to visualize branching morphogenesis and patterning of peripheral nerves and blood vessels, as well as immune cell distribution.

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching ...

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ovarian fitness is often assessed by visualizing and quantifying follicles and oocytes. Because the ovary is an opaque three-dimensional tissue, traditional approaches require laboriously slicing the tissue into numerous serial sections in order to visualize cells throughout the entire organ. Furthermore, because quantification by this method typically entails scoring only a subset of the sections separated by the approximate diameter of an oocyte, it is prone to inaccuracy. Here, a protocol is described that instead utilizes whole organ tissue clearing and immunofluorescence staining of mouse ovaries to visualize follicles and oocytes. Compared to more traditional approaches, this protocol is advantageous for visualizing cells within the ovary for numerous reasons: 1) the ovary remains intact throughout sample preparation and processing; 2) small ovaries, which are difficult to section, can be examined with ease; 3) cellular quantification is more readily and accurately achieved; and 4) the whole organ imaged.

Here, we present a protocol to quantify follicles in cultured ovaries of young mice without serial sectioning. Using whole organ immunofluorescence and tissue clearing, physical sectioning is replaced with optical sectioning. This method of sample preparation and visualization maintains organ integrity and facilitates automated quantification of specific cells.

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...