We thank members of the Muneoka Lab and the Texas Institute for Genomic Medicine (TIGM). This work was supported by Texas A&#38;M University.

All animal use and techniques were in compliance with the standard operating procedures of the Institutional Animal Care and Use Committee of Texas A&#38;M University.
1. Adult Mouse Hind Limb Distal P3 Amputation
<ol>
	<li>Anesthetize an 8 to 12 week-old CD-1 mouse (Table of Materials) using isoflurane gas in oxygen; initially anesthetize at 3% in a chamber, followed by 2% isoflurane supplied by a nosecone over the duration of the surgery. Apply ophthalmic ointment on eyes to prevent dryness while under anesthesia. 
	NOTE: The adult P3 amputation studies standardized in our lab are performed on 8 to 12 week-old mice, and the regeneration response is conserved in all tested strains.</li>
	<li>Under a 10x dissection microscope, sterilize the digits of the hind limb with surgical Povidone-iodine and 70% ethanol. Trim hair away from the surgical site using micro-scissors. Apply 1 &#181;L of topical Bupivacaine locally to anesthetize the surgical site.</li>
	<li>Amputate the distal tip of the terminal phalanx (P3) on digits 2 and 4 of each hind limb using a sterile #10 scalpel (Figure 1A). Amputation must be performed under aseptic conditions, including sterilization of surgical instruments. Under the dissection microscope, gently splay the hind paw to expose the medial digit surface. Hold the scalpel at an angle parallel to the fatpad to perform the amputation shown in Figure 1B.&#160;Apply 1 &#181;L of topical Bupivacaine to the amputation site. 
	NOTE: Amputation transects the nail organ, dermis, the P3 bone, vasculature, and nerves, but does not transect the marrow cavity or the digit fat pad. If bleeding is observed, apply direct pressure using a sterile cotton tip applicator.</li>
	<li>The standard P3 distal amputation plane removes approximately 15%&#8211;20% of the P3 bone volume21. MicroCT scanning (see below) can be performed to confirm the correct amputation length.</li>
	<li>Return the mouse to a clean cage and allow the wound to heal without wound dressing. Monitor the mouse for 3 days to ensure the mouse returns to normal activity.</li>
</ol>
2. Digit Collection and Tissue Preparation
<ol>
	<li>Euthanize the mouse using carbon dioxide (CO2) in a closed chamber, followed by cervical dislocation.</li>
	<li>Use a scalpel to sever the digit mid-way through the adjacent bone segment, the middle phalanx (P2) bone, at approximately the second ventral fat pad indent. Transfer the digit(s) to a 20 mL scintillation vial containing 10 mL fresh buffered zinc formalin fixative, an 18% formaldehyde solution (see Table of Materials). Fixative volume should be at least 20x the volume of the tissue present. 
	NOTE: Digits are collected at various timepoints after amputation to investigate the full regeneration response. Generally, for early blastema formation, bone histolysis, and wound closure the collection timepoints are 4&#8211;8 days post amputation (DPA). To investigate the early osteogenic blastema the collection timepoints are 9 and 10 DPA, and to visualize continued bone regeneration and mineralization, the collection timepoints are 14, 21, and 28 DPA. Unamputated digits are collected as well.</li>
	<li>Fix the digit for 24&#8211;48 h at room temperature with gentle mixing on a shaker.</li>
	<li>Remove the fixative and wash the digit 2x for 5 min in 5 mL of 1x phosphate buffered saline (PBS) at room temperature.</li>
	<li>Place 10 mL of fresh Decalcifier I (see Table of Materials), a 10% formic acid solution, in the scintillation vial and gently mix on a shaker for 2 h at room temperature. After 2 h, replace Decalcifier I with fresh solution and decalcify digit overnight at room temperature with gentle mixing on shaker.</li>
	<li>Remove the Decalcifier I and wash the digit 2x for 5 min in 5 mL of 1x PBS at room temperature.</li>
	<li>Process the digit through a graded ethanol series. This process can be performed manually in a 20 mL scintillation vial, using 10 mL of each liquid if less than 40 total digits.
	<ol>
		<li>Begin with 2 immersions in fresh 70% ethanol at room temperature with gentle mixing on a shaker, 1 h each, followed by 2 immersions in fresh 95% ethanol at room temperature with gentle mixing on a shaker, 1 h each.</li>
		<li>Complete the digit dehydration with 2 washes of fresh 100% ethanol at room temperature with gentle mixing on a shaker, 1 h each. The final 100% ethanol wash can be left overnight.</li>
	</ol>
	</li>
	<li>In the same scintillation vial, replace the 100% ethanol with 10 mL of fresh xylenes, and place the vial in a chemical fume hood for 1.5 h at room temperature. Repeat with a fresh wash of xylenes in a chemical fume hood for 1.5 h at room temperature for a total of 2 washes.</li>
	<li>Replace the xylenes with liquid paraffin wax melted to 68 &#176;C, and place the vial in an incubator at 68 &#176;C for 2 h, 2 times. After 4 total h of immersion in paraffin wax, embed the digit for paraffin sectioning. 
	NOTE: Digit processing for histology can be performed in an automated processor, following these same guidelines.</li>
	<li>Section digits at 4&#8211;5 &#181;m thickness using a microtome. Place sectioned samples on adhesive slides, using a 38&#8211;41 &#176;C water bath supplemented with a histological adhesive solution to ensure samples adhere to the slide. Place slides on a 37 &#176;C slide warmer to dry.</li>
</ol>
3. Immunohistochemical Staining of Adult Mouse Digits to Investigate Blastema Formation and Intramembranous Ossification
<ol>
	<li>Heat sectioned slides at 65 &#176;C for 45 min, followed by heating at 37 &#176;C for no less than 15 min to ensure samples adhere to the slide.</li>
	<li>Deparaffinize and rehydrate slides 2x with 5 min washes of xylenes, a graded ethanol series, and submersion in water. Place slides in a 50&#8211;100 mL capacity staining jar and keep immersed in 1x Tris Buffered Saline with Tween 20 (TBST). 
	NOTE: Do not allow samples to dry throughout the staining process. At any TBST step, slides can be incubated up to 2 h.</li>
	<li>Prepare a humidifying chamber. A 1-inch deep covered plastic slide box with moistened tissue paper at the base is sufficient.</li>
	<li>Prepare the heat retrieval solution for Runx2, Osterix (OSX), and Proliferating Cell Nuclear Antigen (PCNA). For the antigen retrieval of the early osteoprogenitor marker, Runx2, prepare a 1x solution of Tris-EDTA, pH 8. For the osteoblast marker, OSX, prepare a 1x solution of citrate buffer, pH 6. PCNA immunostaining can be performed in either solution.</li>
	<li>Prepare Proteinase K antigen retrieval solution for the blastema marker CXCR422 and the endothelial cell marker von Willebrand Factor 8 (vWF) by placing 100 &#956;L of Proteinase K solution per slide in a microcentrifuge tube and heating to 37 &#176;C (approximately 5 min). 
	NOTE: Antigen retrieval for Runx2, OSX, and PCNA is performed using heat retrieval, and CXCR4 and vWF antigen retrieval is performed via proteinase K treatment.</li>
	<li>For antibody retrieval requiring heat, immerse slides in a staining jar in the appropriate antigen retrieval solution. Heat slides to 95 &#176;C for 25 min, ensuring boiling does not occur. Remove staining jar from heat source and allow to cool at room temperature for 35 min.</li>
	<li>For Proteinase K antigen retrieval, lay slides flat in the humidifying chamber, and place 100 &#956;L of pre-warmed Proteinase K on the slide. Cover the slides with a small strip of parafilm to ensure the slides do not become dry. Incubate slides for 12 min at 37 &#176;C.</li>
	<li>Wash slides 3 times for 5 min in 1x TBST in staining jar after antigen retrieval.</li>
	<li>Remove slides from the staining jar and place in humidifying chamber. Place 6&#8211;7 drops of blocking solution (Table of Materials) on the slide, and cover the slide with fresh paraffin film to ensure the slide does not become dry. Incubate slides for 1 h at room temperature.</li>
	<li>Prepare the primary antibodies by diluting the antibodies into antibody diluent (Table of Materials). Vortex each primary antibody solution for 3 s. Primary antibodies derived from different host species may be combined. 
	NOTE: Immunohistochemical staining for Runx2 (rabbit anti-Runx2 antibody, 1:250 dilution, at a final concentration of 4 &#956;g/mL) combined with PCNA (monoclonal mouse anti-PCNA antibody, 1:2,000 dilution, at a final concentration of 0.5 &#956;g/mL), and OSX (rabbit anti-OSX antibody, 1:400 dilution, at a final concentration of 0.125 &#956;g/mL) combined with PCNA is shown in Figure 2. The mouse anti-PCNA antibody used in this study is highly specific and requires no additional blocking steps beyond those outlined in this protocol. Immunohistochemical staining using CXCR4 (rat anti-CXCR4 antibody, 1:500 dilution at a final concentration of 2 &#956;g/mL) and vWF (rabbit anti-human vWF VIII antibody, 1:800 dilution, at a final concentration of 41.25 &#956;g/mL) is shown in Figure 2. Primary antibodies were optimized and tested by our lab under the conditions in this protocol and are listed in the Table of Materials.</li>
	<li>Carefully remove the paraffin film, and gently drain the slide onto tissue paper to remove excess blocking solution. Place 100&#8211;200 &#956;L of primary antibody solution on the slide, replace parafilm cover, and return to the humidifying chamber. Incubate the slides in the closed humidifying chamber overnight at 4 &#176;C. 
	NOTE: Do not let the slides become dry while draining the blocking solution. This step is performed quickly.</li>
	<li>Carefully remove the parafilm, and gently drain the primary antibody solution from the slide. Place the slide in a staining jar containing fresh 1X TBST. Wash with fresh 1x TBST for 5 min 3 times.</li>
	<li>Prepare the secondary antibodies by combining with antibody diluent (Table of Materials). Vortex the secondary antibody solution for 3 s. Secondary antibodies conjugated to different fluorophores derived from different species may be combined. 
	NOTE: Fluorescent secondary antibodies are light sensitive. Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) for Runx2, OSX, and vWF, Alexa Fluor 568-conjugated goat anti-rat IgG (H+L) for CXCR4, and Alexa Fluor 647-conjugated goat anti-mouse IgG (H+L) for PCNA, diluted at 1:500 with a final concentration of 4 &#956;g/mL, were used in Figure 2.</li>
	<li>Place 200 &#956;L of secondary antibody solution on the slide, cover slide with fresh paraffin film, and return to humidifying chamber. Incubate the slides in the closed humidifying chamber for 45 min at room temperature. Ensure the humidifying chamber is closed to avoid light.</li>
	<li>Carefully remove the paraffin film and gently drain the secondary antibody solution from the slide. Place the slide in a staining jar containing fresh 1x TBST. Wash with fresh 1x TBST for 5 min 3x. Avoid light.</li>
	<li>Prepare nuclear stain. Add 20 &#956;L of DAPI (stock DAPI solution is 5 mg/mL in H2O) to 200 mL of 1x PBS and shake vigorously to ensure homogeneity. Immerse slides for 5 min in DAPI-PBS solution. Avoid light.</li>
	<li>Wash slides in dH2O for 3 min. Decant the water and allow the slides to air-dry or gently aspirate the water from the slide, ensuring that the samples are not disturbed while vacuuming. Avoid light.</li>
	<li>Carefully coverslip dry slides using 100 &#956;L of anti-fade mounting medium (Table of Materials); avoid the formation of air bubbles on the slide during the mounting step. Store the slides flat and allow the mounting medium to dry overnight at room temperature in a light-proof container. After the mounting medium is dry, store slides flat in a light-proof container in 4 &#176;C until ready to view. 
	NOTE: If unacceptable levels of air bubbles are present after mounting, the mounted slide can be gently placed in 1x PBS, the coverslip can be removed, and once the slide is rinsed again in water and re-dried, can be re-mounted.</li>
</ol>
4. Microscopy and Image Analysis
NOTE: Imaging and analysis using a fluorescence deconvolution microscope and associated software, equipped with 3 fluorescent filters (to visualize Alexa Fluor 488, 568, and 647 nm signals), plus DAPI (419 nm) is used in this experiment.
<ol>
	<li>Image the slide at 10x magnification to capture the entire blastema region. 
	NOTE: Proliferating osteoblast primary antibody detection utilizes secondary antibodies (Alexa Fluor 488 and 647) with non-overlapping emission spectra to minimize incorrect identification of co-labeled cells. Background subtraction of autofluorescent signal can be performed using the 568 nm filter to ensure integrity of the 488 or 647 signals. Blastema primary antibody detection utilizes secondary antibodies (Alexa Fluor 488 or 568). Background subtraction of autofluorescent signal can be performed with the 568 filter if using the 488-conjugated antibody, and 488 filter is using the 568-conjugated antibody. Autofluorescent signal is typically associated with the nail plate and erythrocytes throughout the digit, and is observed in the 488, 568, and 647 filters.</li>
</ol>
5. Sequential In Vivo Microcomputed Tomography (&#956;CT)
<ol>
	<li>Regenerating digits of the same animal scanned at 1 day prior to amputation (unamputated), 1 DPA, and at various time points until complete regeneration at 28 DPA using the vivaCT 40 (Table of Materials) is performed in this experiment and shown in Figure 3A.</li>
	<li>Outfit the &#956;CT with tubing to enable oxygen flow into and out of the &#956;CT animal chamber, ensuring that the out-flowing oxygen is equipped with an activated charcoal filter to trap the isoflurane. 
	NOTE: Ensure that the &#956;CT animal chamber is tightly enclosed; in this way, an animal nose-cone is not required for supplying isoflurane to the mouse during the scan.</li>
	<li>Prepare the &#956;CT scanning parameters. Digits are scanned at a voxel size of 10.5 &#956;m, at 55 kVp, 145 uA with 1000 projections per 180&#176; using continuous rotation, and with an integration time of 200 msec, resulting in a maximum of 213 slices per scan. Scans performed in this experiment are approximately 9 min long. A decreased electric current can be compensated with proportionally increased integration time but will result in longer scan times.</li>
	<li>Anesthetize the adult mouse using isoflurane; initially anesthetize at 3% in a chamber, followed by 1.5% isoflurane supplied to the &#181;CT animal chamber over the duration of the scan. Gently place the mouse in the &#181;CT animal chamber with the hindlimb digits arranged in close association, flat, ventral side-up with left paw on left side and right paw on right side on the scanning platform, followed by gently securing the digits in place using surgical tape. Apply ophthalmic ointment on eyes to prevent dryness while under anesthesia.</li>
	<li>Return the mouse to a clean cage and monitor the mouse until awake. Continue scanning the same mouse at bi-weekly to weekly time points to visualize the entire bone regeneration response.</li>
	<li>After scanning, allow up to several hours for &#956;CT image reconstruction to occur. Using the software provided with the &#956;CT, convert the files to a series of dicom files; each dicom file will correspond to one slice of the scan, therefore, if 213 slices have been scanned, 213 dicom files will be generated and uploaded to the company&#8217;s server.</li>
	<li>Download the dicom series in a single folder using a batch file downloader in a web browser.</li>
	<li>Download the BoneJ plugin23 and drag the file into the ImageJ plugin folder. In ImageJ, open the dicom file series as a stack by dragging and dropping the folder to the ImageJ bar. A 16-bit image stack displaying all the gray values will be opened. To display bone only, perform image thresholding (Image &#62; Adjust &#62; Threshold). 
	NOTE: When viewing ImageJ files, the ImageJ output will correspond to an inverted image of the digits; i.e., digits arranged in the scanning platform ventral side-up, with left paw on left side of platform and right paw on right side of platform, will appear as dorsal side-up, right paw on left side and left paw on right side of the ImageJ image stack.</li>
	<li>Once the threshold dialog box is opened, slide the bottom bar, corresponding to the upper threshold, to the far right, and adjust the top bar, corresponding to the lower threshold, until only the bone is highlighted red. The lower threshold value will be approximately 10,000&#8211;13,000 at a maximum signal intensity of 32,767. Click apply, and in the new dialog box click black background. Click OK.</li>
	<li>An 8-bit image displaying black and white values will be generated, with white corresponding to bone. Select the P3 bone by drawing a rectangle around it, and duplicate the stack. Generate a 3D image (Plugins &#62; 3D Viewer). To crop any unnecessary bone from the image, click the freehand selection tool, and circle the bone to be deleted. Right click, and select Fill Selection to delete bone. 
	NOTE: The steps described above apply to ImageJ 1.52h, Java 8 and may differ slightly when using a different version of ImageJ. For thresholding, we recommend the determination of an optimal value and using that value consistently.</li>
	<li>Quantify bone volume from the 3D rendering by using the BoneJ Volume Fraction Plugin (Plugins &#62; BoneJ &#62; Volume Fraction). A result window will appear, and bone volume (BV) will be displayed in mm3.</li>
	<li>Quantify bone length from the 3D rendering by using the ImageJ multipoint tool. 
	NOTE: Bone length is dynamic over the course of P3 regeneration; the length decreases between 7-10 DPA associated with osteoclast-mediated bone degradation11, and is followed by a period of distal bone regrowth between 14-28 DPA (Figure 3B). Length is measured from the central base of the P2/P3 joint to the farthest point of mineralization at the distal bone apex, but does not include degraded bone that has been completely detached (Figure 3B). BoneJ allows for the complete 3D assessment of the bone architecture; thus, the angle in which the bone is scanned will not alter the ability to measure length.</li>
	<li>Capture an image of the 3D rendering by clicking View, and take snapshot. Save image as a tif or jpeg file.</li>
</ol>

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The authors have nothing to disclose.

This protocol describes a standardized procedure of adult mouse distal P3 amputation, fluorescent immunohistochemical staining to visualize and investigate blastema formation and intramembranous ossification, and sequential in-vivo &#181;CT scanning to identify bone morphological, volume, and length changes post amputation. P3 amputation is a unique, procedurally simple, and reproducible model to analyze a pro-regenerative wound environment that triggers blastema formation. Furthermore, the P3 digit model offers numerous...

Mammals, including humans and mice, have the capacity to regenerate the tips of their digits after distal amputation of the terminal phalanx (P3)1,2,3. In mice, the regeneration response is amputation-level-dependent; increasingly proximal digit amputations display a progressively attenuated regenerative response until complete regenerative failure at amputations transecting and proximal to the P3 nail matrix4,5,6,7,8. P3 regeneration is mediated by the formation of a blastema, defined as a population of proliferating cells that undergo morphogenesis to regenerate the amputated structures9. The formation of a blastema to regenerate the structures lost by amputation, a process termed epimorphic regeneration, distinguishes the multi-tissue-level P3 regeneration response from traditional tissue repair after injury6,10. P3 regeneration is a reproducible and procedurally simple model to investigate complex regenerative processes including wound healing11,12, bone histolysis11,12, revascularization13, peripheral nerve regeneration14, and blastemal conversion to bone via intramembranous ossification15.
Previous studies using immunohistochemistry have demonstrated that the blastema is heterogeneous, avascular, hypoxic, and highly proliferative11,13,15,16. Following distal P3 amputation, the early blastema is initially associated with the P3 periosteum and endosteum and is characterized by robust proliferation and nascent osteogenesis adjacent to the bone surface15. Subsequent to bone degradation and wound closure, the heterogeneous blastema is formed by the merging of periosteal and endosteal-associated cells, followed by the differentiation of blastemal components including bone via intramembranous ossification15.
Bone repair in response to injury typically occurs by endochondral ossification, i.e. via an initial cartilaginous callus that forms a template for subsequent bone formation17,18. Long bone intramembranous ossification, i.e., bone formation without a cartilaginous intermediate, is commonly induced using complex distraction devices or surgical fixation19,20. The digit regeneration response is a pre-clinical model that offers advantages over conventional intramembranous ossification models: 1) it does not require external or internal fixation post injury to stimulate intramembranous ossification, 2) it is performed using 4 digits from each animal, thus maximizing samples while minimizing animal use, and 3) sequential in vivo microcomputed tomography (&#956;CT) analysis can be performed with ease and speed.
In the present study, we show the standardized P3 amputation plane to achieve a reproducible and robust regeneration response. Additionally, we demonstrate an optimized fluorescence immunohistochemistry protocol using paraffin sections to visualize blastema formation, revascularization in the context of regeneration, and blastemal conversion to bone via intramembranous ossification. We also demonstrate the use of sequential in-vivo &#956;CT to identify changes in bone morphology, volume, and length in the same digit over the course of regeneration. The goal of this protocol is to investigate mammalian blastema formation after amputation and to demonstrate 2 techniques, fluorescence immunohistochemistry and sequential in vivo &#956;CT, for the study of intramembranous bone regeneration.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Protein Block Serum Free</td>
			<td>DAKO</td>
			<td>X0909</td>
			<td>Ready to use</td>
		</tr>
		<tr>
			<td>Mouse anti-PCNA antibody</td>
			<td>Abcam</td>
			<td>ab29</td>
			<td>1:2000 dilution</td>
		</tr>
		<tr>
			<td>Rat anti-CXCR4 antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB21651</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Rabbit anti-human vWF XIII antibody</td>
			<td>DAKO</td>
			<td>A0082</td>
			<td>1:800 dilution</td>
		</tr>
		<tr>
			<td>Rabbit anti-osterix, SP7 antibody</td>
			<td>Abcam</td>
			<td>ab22552</td>
			<td>1:400 dilution</td>
		</tr>
		<tr>
			<td>Rabbit anti-Runx2 antibody</td>
			<td>Sigma-Aldrich Co.</td>
			<td>HPA022040</td>
			<td>1:250 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647-conjugated goat anti-mouse IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A21235</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568-conjugated goat anti-rat IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A11077</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Prolong Gold antifade reagent</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td>Ready to use</td>
		</tr>
		<tr>
			<td>Surgipath Decalicifier 1</td>
			<td>Leica Biosystems</td>
			<td>3800400</td>
			<td>Ready to use</td>
		</tr>
		<tr>
			<td>Z-Fix, Aqueous buffered zinc formalin fixative</td>
			<td>Anatech LTD</td>
			<td>174</td>
			<td>Ready to use</td>
		</tr>
		<tr>
			<td>CD-1 Female Mouse</td>
			<td>Envigo</td>
			<td>ICR(CD-1)</td>
			<td>8-12-weeks-old</td>
		</tr>
		<tr>
			<td>vivaCT 40</td>
			<td>SCANCO Medical</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

adult mouse digit amputation and regeneration: a simple model to investigate mammalian blastema formation and intramembranous ossification

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic regeneration, which involves blastema formation and intramembranous ossification analyzed by fluorescence immunohistochemistry and sequential in-vivo microcomputed tomography (&#956;CT). Mammalian regeneration is restricted to amputations transecting the distal region of the terminal phalanx (P3); digits amputated at more proximal levels fail to regenerate and undergo fibrotic healing and scar formation. The regeneration response is mediated by the formation of a proliferative blastema, followed by bone regeneration via intramembranous ossification to restore the amputated skeletal length. P3 amputation is a preclinical model to investigate epimorphic regeneration in mammals, and is a powerful tool for the design of therapeutic strategies to replace fibrotic healing with a successful regenerative response. Our protocol uses fluorescence immunohistochemistry to 1) identify early-and-late blastema cell populations, 2) study revascularization in the context of regeneration, and 3) investigate intramembranous ossification without the need for complex bone stabilization devices. We also demonstrate the use of sequential in vivo &#956;CT to create high resolution images to examine morphological changes after amputation, as well as quantify volume and length changes in the same digit over the course of regeneration. We believe this protocol offers tremendous utility to investigate both epimorphic and tissue regenerative responses in mammals.

Adult mouse regenerating P3 digits at 6/7 DPA (Figure 2A-D), 9 DPA (Figure 2E-H), and 10 DPA (Figure 2I-L) were immunostained with antibodies to Runx2, OSX, and PCNA to visualize intramembranous bone regeneration, and immunostained with antibodies to CXCR4 and vWF to visualize blastema formation. Representative &#956;CT renderings of digits scanned prior to amputation and at various...

Watch this Scientific Journal Video about Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification at JoVE.com

Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification

Here, we present a protocol of adult mouse terminal phalanx amputation to investigate mammalian blastema formation and intramembranous ossification, analyzed by fluorescent immunohistochemistry and sequential in-vivo microcomputed tomography.

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic ...

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Developmental Biology

8.6K Views.  Texas A&M University. Here, we present a protocol of adult mouse terminal phalanx amputation to investigate mammalian blastema formation and intramembranous ossification, analyzed by fluorescent immunohistochemistry and sequential in-vivo microcomputed tomography.

Video: Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

Building Finite Element Models to Investigate Zebrafish Jaw Biomechanics

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical strain. Skeletal joints are subjected to dynamic mechanical loading. Finite element analysis (FEA) is a computational method, frequently used in engineering that can predict how a material or structure will respond to mechanical input. By dividing a whole system (in this case the zebrafish jaw skeleton) into a mesh of smaller 'finite elements', FEA can be used to calculate the mechanical response of the structure to external loads. The results can be visualized in many ways including as a 'heat map' showing the position of maximum and minimum principal strains (a positive principal strain indicates tension while a negative indicates compression. The maximum and minimum refer the largest and smallest strain). These can be used to identify which regions of the jaw and therefore which cells are likely to be under particularly high tensional or compressional loads during jaw movement and can therefore be used to identify relationships between mechanical strain and cell behavior. This protocol describes the steps to generate Finite Element models from confocal image data on the musculoskeletal system, using the zebrafish lower jaw as a practical example. The protocol leads the reader through a series of steps: 1) staining of the musculoskeletal components, 2) imaging the musculoskeletal components, 3) building a 3 dimensional (3D) surface, 4) generating a mesh of Finite Elements, 5) solving the FEA and finally 6) validating the results by comparison to real displacements seen in movements of the fish jaw.

Finite Element Analysis is a frequently used tool to investigate the mechanical performance of structures under load. Here we apply its use to modeling the biomechanics of the zebrafish jaw.

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical ...

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 ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

Planarians are flatworms that are extremely efficient at regeneration. They owe this ability to a large number of stem cells that can rapidly respond to any type of injury. Common injury models in these animals remove large amounts of tissue, which damages multiple organs. To overcome this broad tissue damage, we describe here a method to selectively remove a single organ, the pharynx, in the planarian Schmidtea mediterranea. We achieve this by soaking animals in a solution containing the cytochrome oxidase inhibitor sodium azide. Brief exposure to sodium azide causes extrusion of the pharynx from the animal, which we call &#34;chemical amputation.&#34;&#160;Chemical amputation removes the entire pharynx, and generates a small wound where the pharynx attaches to the intestine. After extensive rinsing, all amputated animals regenerate a fully functional pharynx in approximately one week. Stem cells in the rest of the body drive regeneration of the new pharynx. Here, we provide a detailed protocol for chemical amputation, and describe both histological and behavioral methods to assess successful amputation and regeneration.

Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

The planarian Schmidtea mediterranea is an excellent model for studying stem cells and tissue regeneration. This publication describes a method to selectively remove one organ, the pharynx, by exposing animals to the chemical sodium azide. This protocol also outlines methods for monitoring pharynx regeneration.

Planarians are flatworms that are extremely efficient at regeneration. They owe this ability to a large number of stem cells that can rapidly respond to ...

Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult liver regeneration after 70% partial hepatectomy results in hepatocyte hypertrophy with some replication in remaining lobes with restoration of metabolic activity, but with permanent loss of the injured lobe&#39;s morphology and architecture. Here, we detail a new surgical method in the neonate that leaves a physiologic environment conducive to regeneration. This model involves amputation of the left lobe apex and a subsequent conservative management regimen, and lacks the necessity for ligation of major liver vessels or chemical injury, leaving a physiologic environment where regeneration may occur. We extend this protocol to amputations on juvenile (P7-14) mice, during which the injured liver transitions from organ regeneration to compensatory growth by hypertrophy. The presented, brief 30 min protocol provides a framework to study the mechanisms of regeneration, its age-associated decline in mammals, and the characterization of putative hepatic stem or progenitors.

Here, we present a new method for partial resection of the left hepatic lobe in neonatal (day 0) mice. This new protocol is suitable for studying acute liver injury and injury response in the neonatal setting.

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...