The authors would like to thank Sabine Stumpp for excellent technical support and Lidia Gr&#246;sser for assistance in the surgeries. This research was funded by the Austrian Science Fund [Hertha Firnberg Fellowship number T-1219], ERC [Advanced Grant, 742046 RegGeneMems], DFG [CRC 1444].

The following procedure was performed with approval from the Magistrate of Vienna (GZ: MA 58-65248-2021-26). 5-8 years old, &#8805; 20 cm snout-to-tail tip (snout to the tip of the tail) long axolotls (Ambystoma mexicanum) were used for fracture surgery and amputations. Both males and females were used for the surgeries. Axolotls were bred in the Research Institute of Molecular Pathology facility. Pain and risk of infections were managed with proper analgesics and antibiotics to ensure a successful outcome. The reagents and equipment used for the study are listed in the Table of Materials.
1. Animal preparation
<ol>
	<li>Bath the animal in 0.03% benzocaine solution for about 15-20 min until full sedation is reached and there is no reflexive movement upon limb touching with tweezers.</li>
	<li>Place the animal with the ventral side down on wet paper towels soaked in 0.03% benzocaine solution and cover it with benzocaine-soaked paper towels. The skin of aquatic animals, such as axolotls, is sensitive to drying, and thus, it is essential to cover the body surface to prevent dehydration and ensure cutaneous (skin) respiration.</li>
	<li>Stretch out the hindlimb to be operated using ring forceps. Do not apply disinfection reagents, such as ethanol, since axolotl skin is sensitive to chemicals and easily irritated. Instead, use 0.7x PBS (A-PBS) with 50 U/mL penicillin and 20 &#181;g/mL streptomycin for cleaning the limb and later on for bone irrigation upon sawing. 
	NOTE: The infection is generally not a concern for the surgeries performed on axolotls. However, due to the aquatic nature of these animals and the sutures placed on the skin surface, we recommend the use of antibiotics to prevent any contamination of the surgical site.</li>
</ol>
2. Surgery
NOTE: Sterilize all surgical tools. Common sterilization methods such as heat sterilization, autoclaving, and washing in 70% ethanol, followed by thorough removal of the alcohol remnants, are suitable for this purpose. If operating on multiple animals, sterilize tools in between using a hot bead sterilizer or 70% ethanol.
<ol>
	<li>Make a lateral longitudinal incision (1.5-2 cm) with a scalpel above the femur bone spanning the whole thigh in the upper hind limb. In order to do this, palpate the bone before cutting the skin.</li>
	<li>Carefully displace the muscles and nerves from the surgery site without cutting. Use bowed forceps to do it efficiently.</li>
	<li>Gently put bowed forceps under the femur to expose it for the surgery.</li>
	<li>Place a rigid 7.75 mm 4-hole fixator plate along with the femur diaphysis, avoiding touching the joints, and secure it in the aligned position with forceps.</li>
	<li>Use four 2 mm titanium screws to attach the bone to the plate. 
	NOTE: The screws used in this protocol have a complex design and consist of 4 parts: the main part (will be screwed into the bone), screw head (allows removal of the screws and plate using the square box wrench), narrower neck (used as a breaking point once the screw is tightened in the bone) and a screw handle (used for attaching to the screwdriver and the saw-guiding device).</li>
	<li>The order of screw attachment is important. Start with the inner screws first, and then the two outer screws to ensure the plate is aligned with the bone axis. Use a manual drill to create the first hole in the bone for easy insertion of the screw, followed by 1st screw placing. Drill in the middle of the bone circumference to avoid thinner bone on one side, which may result in spontaneous bone fracture. Use irrigation with 0.7x PBS + 1% Pen/Strep during drilling. Do not break off the handle of the 1st (optional: 1st and 2nd) screw(s).</li>
	<li>Apply the saw-guiding device onto the 1st (optional: 1st and 2nd ) screw(s) and align it with the bone and plate. 
	NOTE: In this protocol, a plate, screws, a saw-guiding device, and a saw are provided by the same manufacturer and optimized to fit each other. The saw-guiding device can come in different sizes to be compatible with different plates and saw sizes.</li>
	<li>Use the saw-guiding device to drill and insert the rest of the screws. Ensure alignment of the plate with the bone. Break off the handles of the screws.</li>
	<li>Place a piece of plastic film (6-7 mm by 4-5 cm), sterilized by wiping with 70% ethanol and then autoclaved or heat sterilized (140 &#176;C for 4 hours), under the femur to prevent soft tissue damage during the osteotomy process. 
	NOTE: For this purpose, a piece of plastic film, cut off a bag for heat sterilization can be used.</li>
	<li>Place Gigly wire saw between bone and protection film.</li>
	<li>Cut bone using the 0.66 mm Gigly wire saw, creating a single 0.7 mm cut in the femur. Use constant irrigation with 0.7x PBS + 1% Pen/Strep during sawing to minimize tissue damage and friction.</li>
	<li>Remove the saw, and saw guide and use a screwdriver to break off the screw handles from the screws.</li>
	<li>Remove the protection film, and irrigate the surgery site with 0.7x PBS + 1% Pen/Strep.</li>
	<li>Cover the top of the plate and screws with sterile bone bee wax to protect skin and muscles from irritation by the edges of the screws.</li>
	<li>Place muscles and skin on top of the bone bee wax.</li>
	<li>Close the incision site with a 7.0 synthetic (polypropylene/polyethylene) suture using simple interrupted stitches. Synthetic suture is used to minimize contamination with water-born bacteria and fungi.</li>
</ol>
3. Postoperative management
<ol>
	<li>To reawaken the animal, place it in a tank with fresh artificial pond water, supplemented with 50 U/mL penicillin, 20 &#181;g/mL streptomycin, and analgetic Butorphanol (0.5 mg/L water).</li>
	<li>Observe the animal to start moving gills, making steps, and swimming - usually within 1 h post-surgery.</li>
	<li>Keep the animal for 3 days in artificial pond water with 50 U/mL penicillin and 20 &#181;g/mL streptomycin before returning it to the holding tank. To ensure analgesia, add Butorphanol (0.5 mg/L water).</li>
	<li>Ensure that the sutures remain in place and proper wound healing occurs.</li>
</ol>

Using Plate Fixation to Study Bone Healing and Limb Regeneration in Axolotls

<ol>
	<li>Amamoto, R., 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=Adult+axolotls+can+regenerate+original+neuronal+diversity+in+response+to+brain+injury.">Adult axolotls can regenerate original neuronal diversity in response to brain injury.</a> Elife. 5, 13998 (2016).</li><li>Butler, E. G., Ward, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+the+spinal+cord+following+ablation+in+urodele+larvae.">Reconstitution of the spinal cord following ablation in urodele larvae.</a> J Exp Zool. 160 (1), 47-65 (1965).</li><li>Echeverri, K., Tanaka, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectoderm+to+mesoderm+lineage+switching+during+axolotl+tail+regeneration.">Ectoderm to mesoderm lineage switching during axolotl tail regeneration.</a> Science. 298 (5600), 1993-1996 (2002).</li><li>Vargas-Gonzalez, A., Prado-Zayago, E., Leon-Olea, M., Guarner-Lans, V., Cano-Martinez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+regeneration+in+Ambystoma+mexicanum+after+surgical+injury.">Myocardial regeneration in Ambystoma mexicanum after surgical injury.</a> Arch Cardiol Mex. 75 (3), S321-S329 (2005).</li><li>Vieira, W. A., Wells, K. M., McCusker, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advancements+to+the+axolotl+model+for+regeneration+and+aging.">Advancements to the axolotl model for regeneration and aging.</a> Gerontology. 66 (3), 212-222 (2020).</li><li>Song, F., Li, B., Stocum, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphibians+as+research+models+for+regenerative+medicine.">Amphibians as research models for regenerative medicine.</a> Organogenesis. 6 (3), 141-150 (2010).</li><li>McCusker, C., Bryant, S. V., Gardiner, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+axolotl+limb+blastema:+Cellular+and+molecular+mechanisms+driving+blastema+formation+and+limb+regeneration+in+tetrapods.">The axolotl limb blastema: Cellular and molecular mechanisms driving blastema formation and limb regeneration in tetrapods.</a> Regeneration (Oxf). 2 (2), 54-71 (2015).</li><li>Einhorn, T. A., Gerstenfeld, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fracture+healing:+Mechanisms+and+interventions).">Fracture healing: Mechanisms and interventions).</a> Nat Rev Rheumatol. 11 (1), 45-54 (2015).</li><li>Polikarpova, 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=The+specialist+in+regeneration-the+Axolotl-a+suitable+model+to+study+bone+healing.">The specialist in regeneration-the Axolotl-a suitable model to study bone healing.</a> NPJ Regen Med. 7 (1), 35 (2022).</li><li>Chen, X., 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=The+axolotl+fibula+as+a+model+for+the+induction+of+regeneration+across+large+segment+defects+in+long+bones+of+the+extremities.">The axolotl fibula as a model for the induction of regeneration across large segment defects in long bones of the extremities.</a> PLoS One. 10 (6), e0130819 (2015).</li><li>Cosden-Decker, R. S., Bickett, M. M., Lattermann, C., MacLeod, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+analysis+of+intra-articular+interzone+tissue+in+axolotl+salamanders.">Structural and functional analysis of intra-articular interzone tissue in axolotl salamanders.</a> Osteoarthritis Cartilage. 20 (11), 1347-1356 (2012).</li><li>Williams, J. N., Li, Y., Valiya Kambrath, A., Sankar, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+generation+of+closed+femoral+fractures+in+mice:+A+model+to+study+bone+healing.">The generation of closed femoral fractures in mice: A model to study bone healing.</a> J Vis Exp. (138), e58122 (2018).</li><li>Cheung, K. M., 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=An+externally+fixed+femoral+fracture+model+for+mice.">An externally fixed femoral fracture model for mice.</a> J Orthop Res. 21 (4), 685-690 (2003).</li><li>Jiang, S., Knapstein, P., Donat, A., Tsitsilonis, S., Keller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+protocol+for+a+standardized,+femoral+osteotomy+model+to+study+fracture+healing+in+mice.">An optimized protocol for a standardized, femoral osteotomy model to study fracture healing in mice.</a> STAR Protoc. 2 (3), 100798 (2021).</li><li>Matthys, R., Perren, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internal+fixator+for+use+in+the+mouse.">Internal fixator for use in the mouse.</a> Injury. 40, S103-S109 (2009).</li><li>Manassero, M., 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=Establishment+of+a+segmental+femoral+critical-size+defect+model+in+mice+stabilized+by+plate+osteosynthesis.">Establishment of a segmental femoral critical-size defect model in mice stabilized by plate osteosynthesis.</a> J Vis Exp. (116), e52940 (2016).</li><li>Gunderson, Z. J., Campbell, Z. R., McKinley, T. O., Natoli, R. M., Kacena, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+review+of+mouse+diaphyseal+femur+fracture+models.">A comprehensive review of mouse diaphyseal femur fracture models.</a> Injury. 51 (7), 1439-1447 (2020).</li><li>Riquelme-Guzman, 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=Postembryonic+development+and+aging+of+the+appendicular+skeleton+in+Ambystoma+mexicanum.">Postembryonic development and aging of the appendicular skeleton in Ambystoma mexicanum.</a> Dev Dyn. 251 (6), 1015-1034 (2022).</li><li>Gentz, E. 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=Medicine+and+surgery+of+amphibians.">Medicine and surgery of amphibians.</a> ILAR J. 48 (3), 255-259 (2007).</li><li>Lang, 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=Collagen+I-based+scaffolds+negatively+impact+fracture+healing+in+a+mouse-osteotomy-model+although+used+routinely+in+research+and+clinical+application.">Collagen I-based scaffolds negatively impact fracture healing in a mouse-osteotomy-model although used routinely in research and clinical application.</a> Acta Biomater. 86, 171-184 (2019).</li></ol>

The authors declare no competing interests.

The currently described method of femur plate fixation and osteotomy allows for its application in aquatic animals, such as Ambystoma mexicanum (axolotl). This surgical method was recently used to compare fracture healing and limb regeneration in axolotls to fracture healing in mice9. As in mice, a 4-hole fixator plate can be attached to the bone with self-breaking screws, and a Gigly saw can be used to create a fracture of uniform size15. Plate fixation facilitate...

The axolotl (Ambystoma mexicanum) is an important model for organ regeneration, including the tail, spinal cord, brain, heart, gills, and limbs1,2,3,4,5. Detailed studies of axolotl limb regeneration uncovered mechanisms of cell dedifferentiation and the formation of a stem cell pool, blastema, at the amputation site. Due to the ability of the blastema cells to reconstruct all missing limb parts, including a patterned skeleton6,7, the axolotl appears to be an attractive model organism for bone healing studies. Recently, several studies focused more on bone biology in axolotls, describing skeletal morphology, cellular composition, and ossification dynamics.
It was found in mammals that the bone healing process in long bones occurs via endochondral ossification and consists of several stages: hematoma, granulation tissue, and soft callus formation, callus ossification into hard callus and woven bone, and bone remodeling8. A recent study has shown that similar stages can be observed in axolotl bone healing9.
Until now, axolotl fractures were studied in a non-stabilized system, whereby bone is simply cut with iridectomy scissors. The large fractures were created in the zeugopod, where osteotomy is performed on one of the bones, whereas the other serves as support10,11. In contrast, fractures are routinely studied in mammals, including rats and mice, using reliable fixation systems, such as intramedullary pin and bone-aligning plates, to control fracture size and ensure bone alignment.
Thus, the method aims to ensure stabilized and uniform fixation of the axolotl femur prior to osteotomy. In order to make axolotl studies more comparable to mammals, including mice and humans, intramedullary pin12, external plate fixator13,14, and internal bone aligning plate15,16,17&#160;fixation were considered. The latter was shown to ensure proper bone fixation and allow for creating a gap of a certain size by using one or two cuts with a Gigly saw of a specific diameter. As axolotls represent the aquatic larvae of Ambystoma mexicanum, the external fixator might have caused post-surgical complications due to the open wound and contact with water. As axolotls do not develop secondary ossification centers even until very late in their development (20 years old18), and thus the standard intramedullary nail used in mice might not be prevented from puncturing the epiphyses, a decision was made to apply an internal plate fixation method to large axolotls. In large axolotls, the femur size and degree of ossification resemble that of an adult mouse, thus allowing for mid-diaphyseal osteotomy with titanium plate fixation1.
The fracture gap size largely determines the healing dynamics and outcome. For example, in a mouse, a 0.25 mm stabilized fractures heal mostly through intramembranous&#160;ossification due to their small size and rigid stabilization; a 0.7 mm fracture heals by endochondral ossification, with the formation of a cartilaginous callus around the fracture; large defects, such as 3.5 mm critical-sized defects do not heal completely and thus are used to model bone fracture non-union16. In this study, the plate fixation protocol of the axolotl femur prior to the osteotomy using the example of a 0.7 mm fracture gap was established with the ultimate goal of comparing axolotl bone healing to that of the mouse9.
After osteotomy, the fractures underwent the process of endochondral ossification, albeit slower than in mice, possibly due to the aquatic lifestyle of axolotls and slower cell division rates. In the method presented here, the 0.7 mm gap osteotomy with rigid plate fixation is shown; however, other gap sizes and semi-flexible fixators, as well as plates of different materials, are potentially possible. Overall, the method presented here can be used for standardized bone fixation and will be helpful for studies comparing axolotl limb regeneration to bone healing or studying bone healing in axolotls under different conditions to ensure standardized fracture fixation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.66 mm Gigly wire saw&#160;</td>
			<td>RISystem</td>
			<td>RIS.590.120</td>
			<td></td>
		</tr>
		<tr>
			<td>7.0 Optilene suture&#160;</td>
			<td>Braun</td>
			<td>C3090538</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzocaine&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>E1501</td>
			<td>dilute to 0.03%&#160; prior to using</td>
		</tr>
		<tr>
			<td>Butorphanol (Butomidor 10 mg/mL)</td>
			<td>Richter Pharma AG</td>
			<td>-</td>
			<td>dilute to 0.5 mg/L&#160; prior to using</td>
		</tr>
		<tr>
			<td>Drill bit 0.30 mm</td>
			<td>RISystem</td>
			<td>RIS.590.200</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps - Standard/Inox</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Hand drill</td>
			<td>RISystem</td>
			<td>RIS.390.130</td>
			<td>better to have at least 3 pieces</td>
		</tr>
		<tr>
			<td>Micro CT data analyzer</td>
			<td>Bruker, Billerica, MA, USA</td>
			<td>SkyScan NRecon software</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro CT specimen scanner</td>
			<td>Bruker, Billerica, MA, USA</td>
			<td>SkyScan 1172&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Moria MC31b Iris forceps - smooth, curved, 10 cm</td>
			<td>Fine Science Tools</td>
			<td>11373-12FST</td>
			<td>2 pieces</td>
		</tr>
		<tr>
			<td>MouseFix Drill-&#38;Saw guide 1.75 mm, rigid</td>
			<td>RISystem</td>
			<td>RIS.301.102</td>
			<td></td>
		</tr>
		<tr>
			<td>MouseFix plate 4 hole, rigid</td>
			<td>RISystem</td>
			<td>RIS.401.110</td>
			<td></td>
		</tr>
		<tr>
			<td>MouseFix screw, L =2.00 mm</td>
			<td>RISystem</td>
			<td>RIS.401.100</td>
			<td>need 4 per bone</td>
		</tr>
		<tr>
			<td>Narrow Pattern Forceps</td>
			<td>VWR</td>
			<td>FSCI11002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Ring forceps</td>
			<td>Fine Science Tools</td>
			<td>11103-09</td>
			<td></td>
		</tr>
		<tr>
			<td>scalpel #15</td>
			<td>B Braun, Thermo Fischer Scientific</td>
			<td>5518032</td>
			<td></td>
		</tr>
		<tr>
			<td>Square box wrench 0.50 mm</td>
			<td>RISystem</td>
			<td>RIS.590.111</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile bone wax, 2.5 g</td>
			<td>Ethicon, Johnson &#38; Johnson</td>
			<td>W810</td>
			<td></td>
		</tr>
		<tr>
			<td>Student Fine Scissors - Straight/11.5cm</td>
			<td>Fine Science Tools</td>
			<td>91460-11</td>
			<td></td>
		</tr>
	</tbody>
</table>

stabilizing a femur osteotomy with a plate fixation in ambystoma mexicanum

The axolotl (Ambystoma mexicanum) is a promising model organism for regenerative medicine due to its remarkable ability to regenerate lost or damaged organs, including limbs, brain, heart, tail, and others. Studies on axolotl shed light on cellular and molecular pathways ruling progenitor activation and tissue restoration after injury. This knowledge can be applied to facilitate the healing of regeneration-incompetent injuries, such as bone non-union. In the current protocol, the femur osteotomy stabilization using an internal plate fixation system is described. The procedure was adapted for use in aquatic animals (axolotl, Ambystoma mexicanum). &#8805;20 cm snout-to-tail tip axolotls with fully ossified, mouse-size comparable femurs were used, and special attention was paid to the plate positioning and fixation, as well as to the postoperative care. This surgical technique allows for standardized and stabilized bone fixation and could be useful for direct comparison to axolotl limb regeneration and analogous studies of bone healing across amphibians and mammals.

The surgical procedure described here (Figure 1) lasts between 20 min and 30 min and requires a surgeon and an assistant. Optionally, use a binocular dissection microscope or magnifying glass system.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66648/66648fig01.jpg" /> 
Figure 1: Schematics of the surgical procedure and the experimental setup. (A</s...

Read this Scientific Article Protocol about Author Spotlight: Exploring Regeneration in Axolotls Through Insights in Cellular and Molecular Mechanisms, Bone Healing, and Implications for Human Therapi

Author Spotlight: Exploring Regeneration in Axolotls Through Insights in Cellular and Molecular Mechanisms, Bone Healing, and Implications for Human Therapies

A protocol for femoral osteotomy surgery with the use of internal plate fixation in mature axolotls is presented. The procedure can be used to perform comparative studies on limb regeneration and fracture healing in aquatic amphibians.

Stabilizing a Femur Osteotomy with a Plate Fixation in Ambystoma mexicanum

The axolotl (Ambystoma mexicanum) is a promising model organism for regenerative medicine due to its remarkable ability to regenerate lost or damaged ...

stabilizing-femur-osteotomy-with-plate-fixation-ambystoma

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498 Views.  Research Institute of Molecular Pathology. A protocol for femoral osteotomy surgery with the use of internal plate fixation in mature axolotls is presented. The procedure can be used to perform comparative studies on limb regeneration and fracture healing in aquatic amphibians.

Video: Author Spotlight: Exploring Regeneration in Axolotls Through Insights in Cellular and Molecular Mechanisms, Bone Healing, and Implications for Human Therapies

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

One-channel Cell-attached Patch-clamp Recording

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

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

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

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to ...

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

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

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

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

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that has emerged as an essential model for regeneration studies. Little is known about how revascularization occurs in the context of regeneration in these animals. Here we report a simple method for visualization of the vasculature in axolotl via perfusion of 1,1&#8217;-Dioctadecy-3,3,3&#8217;,3&#8217;-tetramethylindocarbocyanine perchlorate (DiI). DiI is a lipophilic carbocyanine dye that inserts into the plasma membrane of endothelial cells instantaneously. Perfusion is done using a peristaltic pump such that DiI enters the circulation through the aorta. During perfusion, dye flows through the axolotl&#8217;s blood vessels and incorporates into the lipid bilayer of vascular endothelial cells upon contact. The perfusion procedure takes approximately one hour for an eight-inch axolotl. Immediately after perfusion with DiI, the axolotl can be visualized with a confocal fluorescent microscope. The DiI emits light in the red-orange range when excited with a green fluorescent filter. This DiI perfusion procedure can be used to visualize the vascular structure of axolotls or to demonstrate patterns of revascularization in regenerating tissues.

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Using a lipophilic 1,1&#39;-Dioctadecy-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI) staining technique, Ambystoma mexicanum can undergo vascular perfusion to allow for easy visualization of the vasculature.

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that ...

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, chikungunya and Zika. Wolbachia-infected mosquitoes can be released into the field to either reduce population sizes through incompatible matings or to transform populations with mosquitoes that are refractory to virus transmission. For these strategies to succeed, the mosquitoes released into the field from the laboratory must be competitive with native mosquitoes. However, maintaining mosquitoes in the laboratory can result in inbreeding, genetic drift and laboratory adaptation which can reduce their fitness in the field and may confound the results of experiments. To test the suitability of different Wolbachia infections for deployment in the field, it is necessary to maintain mosquitoes in a controlled laboratory environment across multiple generations. We describe a simple protocol for maintaining Ae. aegypti mosquitoes in the laboratory, which is suitable for both Wolbachia-infected and wild-type mosquitoes. The methods minimize laboratory adaptation and implement outcrossing to increase the relevance of experiments to field mosquitoes. Additionally, colonies are maintained under optimal conditions to maximize their fitness for open field releases.

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes infected with Wolbachia are being released into natural populations to suppress the transmission of arboviruses. We describe methods to rear Ae. aegypti with Wolbachia infections in the laboratory for experiments and field release, taking precautions to minimize laboratory adaptation and selection.

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, ...

A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against soilborne fungus Rhizoctonia solani. R. solani is a pathogen of a wide range of plant hosts worldwide. The fungus survives in soils as a saprophyte and grows rapidly on simple water agar media. The plate assay is a rapid method to compare composts for their ability to slow the growth of R. solani. The assay also correlates well with suppression of other soilborne fungal pathogens that survive as saprophytes in soils such as Alternaria early blights, Fusarium wilt, Phytophthora root rot, and Pythium root rot.

Presented is a protocol for a plate competition assay to identify whether a specific compost is likely to contain bacteria and fungi that suppress growth of Rhizoctonia solani.

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against ...

Stabilizing a Femur Osteotomy with a Plate Fixation in Ambystoma mexicanum

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Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Measurement of Cellular Chemotaxis with ECIS/Taxis

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

One-channel Cell-attached Patch-clamp Recording

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

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

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression