This work was supported by the NIH grants K08NS065157 (to T.A.F.) In addition, the Penn Center for Musculoskeletal Disorders, Award Number P30AR050950 from the National Institute of Arthritis, Musculoskeletal and Skin Diseases supported this work (T.A.F. and Steven S. Scherer). Finally, Shriners Pediatric Research Center Seed funding (T.A.F) supported this work. We would like to acknowledge Dr. Young-Jin Son for initially demonstrating the whole mount procedure and Amy A. Kim for her assistance in producing the sketches of Figure 1.

1. Animal Subjects
1.1. Treatment
<ol>
	<li>All animal procedures should be performed with the approval of the local Institution's Animal Care and Ethics Committee and in accordance with the Use and Committee and National Institutes of Health guidelines, with measures taken to minimize pain and discomfort.</li>
	<li>Our mice were housed under temperature-controlled conditions on a 12-hour reverse light and dark cycle, while fed mouse chow and water ad libitum.</li>
	<li>For studies of adult regeneration, mice should be at least 6 weeks of age when the sciatic nerve crush is performed. This age is beyond the time at which pruning of polyneural neuromuscular junctions take place.</li>
	<li>In these experiments, we used 6-8 week old C57BL/6 mice acquired from Charles River. When comparing regeneration after sciatic nerve crush (i.e.: rate of axon growth) mouse strains should be the same, as differences in axonal regeneration have been noted between different inbred strains15,16. If genetically manipulated mice are to be used, littermate controls are most appropriate.</li>
</ol>
1.2. Surgical Preparation
<ol>
	<li>Animals are deeply anesthetized for surgery using a cocktail of ketamine (100 mg/kg) and xylazine (10 mg/kg) via intraperitoneal injection. Each animal also receives a subcutaneous injection of meloxicam (10 mg/kg) to minimize post-operative pain.</li>
	<li>Both hindquarters are carefully shaved using a surgical clippers (Roboz, RC-5903) and depilation is completed with Nair hair removal cream (found at local pharmacy).</li>
	<li>Skin is cleansed using sterile cotton tipped applicators and betadine surgical scrub (Fisher Scientific, 19066452).</li>
	<li>Ophthalmic ointment (Fisher Scientific, 19082795) is applied to the eyes using sterile cotton tipped applicators.</li>
	<li>The mouse is placed on a clean stainless steel plate, under which has been placed a pre-heated homeothermic blanket system (Harvard Apparatus, 507222F). Animal temperature is maintained at 37&deg;C.</li>
	<li>All limbs are taped down, with care taken to position the hind limbs symmetrically so that the knee joint makes a right angle with the body (Figure 1, panel A).</li>
	<li>The surgical field is covered with a sterile drape. All instruments are sterilized by autoclave or hot bead sterilization (Fine Science Tools, 18000-45) and the surgeon wears a mask, gown, and sterile gloves.</li>
</ol>
2. Reproducible Sciatic Nerve Crush
<ol>
	<li>After preparation, a semi-circular incision across midline (Figure 1) is made in the skin. The skin is gently dissected from the underlying musculature, and folded over to remain out of the way during the procedure. It is kept moist using applications of 0.1 mL sterile saline (Hospira, 0409-4888-20) during the procedure.</li>
	<li>Opening the fascial plane between the gluteus maximus and the anterior head of the biceps femoris reveals the sciatic nerve (Figure 1, panel A). For a surgical control, the contralateral sciatic nerve should be exposed and mobilized, but left intact. The gluteal musculature is then re-opposed and sutured using a 6-0 braided silk, non-absorbable sutures (Roboz, SUT-1073-11).</li>
	<li>The experimental sciatic nerve is then exposed in the same fashion, with retractors in place to ease visualization (Figure 1, panel B). The retractors are sterilized prior to use. 
	Note: although retractor systems are commercially available, they are often quite expensive. We were able to make a satisfactory retractor system using inexpensive hardware supplies and insect pins (see Materials section).</li>
	<li>The sciatic nerve is then gently freed from the surrounding connective tissue using iridectomy scissors.</li>
	<li>Using a fine 5/45 (Fine Science Tools, 11251-35) forceps, the nerve is placed on the bottom jaw of a super-fine hemostatic forceps (Fine Science Tools, 13020-12). The three fascicles are sequentially aligned, not on top of each other (Figure 1, inset B). The hemostatic forceps have been engraved with a mark at 1.5 mm from their tip. The outermost portion of the sciatic is placed in line with this mark before crush. This ensures a crush of uniform width, and that the nerve does not extend beyond the jaws of the hemostatic forceps when flattened due to the crushing force. If the nerve extends beyond the forceps tip the nerve will only be partially crushed.</li>
	<li>The crush is made perpendicular to the nerve at 45 mm from the third toe, as measured by a thread that approximates the path of the sciatic nerve. The nerve is crushed once for 15 seconds at 3 clicks of the hemostatic forceps. Care is taken not to stretch the nerve. When the hemostats are re-opened, the entire nerve should be translucent at the crush site.</li>
	<li>A second pair of hemostatic forceps (identical to the first) that has been pre-dipped in powdered carbon (Fisher Scientific, C272-500) is used to mark the crush site. The nerve is crushed at the same crush site for 15 seconds at 3 clicks. No carbon marking should extend beyond the boundary of the initial crush. This is particularly important if precise marking of the crush site is required. Prior to use, carbon powder is sterilized by exposure to UV light for two hours, and thereafter is handled using sterile technique.
	<ol>
		<li>To pre-dip the forceps in carbon but preclude widespread carbon at the surgical site, the forceps are opened in powdered carbon, then gently closed (but not clicked) shut, and the carbon on the outside of the hemostats is wiped off using sterile gauze. The forceps are checked under at least 3x magnification to verify that the crushing surfaces are evenly coated in powdered carbon. If necessary, they are re-dipped and wiped.</li>
	</ol>
	</li>
	<li>The gluteal musculature is re-opposed and sutured in the same way as the contralateral side.</li>
	<li>Finally, the skin incision is closed using 9 mm reflex clips (World Precision Instruments, 500346; Applier: 500345). If 9 mm reflex clips are found to restrict movement, smaller reflex clips or 6-0 sutures (Roboz, SUT-1073-11) can be used instead.</li>
</ol>
3. Post-Operative Care
<ol>
	<li>Following the procedure, animals are placed on a heating pad at 37&deg;C until they show signs of movement.</li>
	<li>They are then moved back to their home cage, where water and food are readily accessible on the floor in the form of Hydrogel and wetted food.</li>
</ol>
4. Semi-Thin Preparation
<ol>
	<li>Following an overdose of pentobarbital (300 mg/kg) the leg musculature is removed to expose the sciatic nerve*. With the nerve remaining in situ, the hindquarter is immersed in 2% paraformaldehyde and 2% gluteraldehyde in 0.1 M phosphate buffer on ice for 30 minutes. 
	*If completing whole mount muscle preparation, muscles are harvested before exposing the sciatic nerve. </li>
	<li>The nerve is carefully removed, with care taken to only handle the proximal end. Then the nerve is post-fixed in the same fixative for three additional hours.</li>
	<li>After fixation the nerve is rinsed three times in 0.1 M phosphate buffer.</li>
	<li>The nerve is immersed in 2% osmium tetroxide in 0.1 M phosphate buffer for one hour.</li>
	<li>The nerve is then dehydrated by sequential immersion in increasingly concentrated ethanol (50%, 70%, 80%, 95%, 100%, 100%, 100%). Each immersion is 15 minutes.</li>
	<li>The dehydrated nerve is incubated twice in propylene oxide for three minutes each.</li>
	<li>Then the nerve is immersed in a 1:1 mixture of propylene oxide and Embed 812 for at least 6 hours (usually overnight).</li>
	<li>Then the nerve is immersed in a 2:1 mixture of propylene oxide and Embed 812 overnight.</li>
	<li>Finally, the nerve is immersed in pure Embed 812 for six hours, then embedded in the appropriate mold and baked at 60&deg;C for 48 hours.</li>
	<li>1.0 &mu;m sections are cut from the distal nerve stump at a set distance from the crush site using an Ultracut UCT ultramicrotome (Leica) with a glass knife and stained with toluidine blue. Thin sections may also be produced for examination of ultrastructure by electron microscopy.</li>
</ol>
5. Whole Mount Muscle Preparation
<ol>
	<li>First mice are sacrificed with an overdose of pentobarbital (300 mg/kg) and the hind limbs are removed at the knees.</li>
	<li>Four muscles in the hind limb are removed for analysis: the tibialis anterior (TA), extensor digitorum longus (EDL), soleus, and peroneus longus.</li>
	<li>All muscles are removed via careful dissection and pinned through their connective tissue to a black sylgard (Fisher Scientific, NC9492579) coated dish. They are rinsed in PBS, and then fixed in 4% paraformaldehyde for 30 minutes.</li>
	<li>The TA is then rinsed in PBS, embedded in O.C.T (Fisher Scientific, 14-373-65), and quickly frozen in a bath of acetone and dry ice.
	<ol>
		<li>It is stored at -80 &deg;C for thin sectioning in case whole mount preparation fails. In general, the TA is too thick for adequate whole mounts.</li>
	</ol>
	</li>
	<li>The EDL, soleus, and peroneus longus are rinsed for 3 x 10 minutes in PBS, placed in 0.1 M glycine* (Fisher Scientific, AC12007-0010; diluted in PBS) for 30 minutes, and rinsed 3 x 10 minutes in PBS again. They are then quenched in ice cold 100% methanol for exactly 5 minutes at -20&deg;C, rinsed 3 x 10 minutes in PBS, and bathed in fluorescently conjugated alpha-bungarotoxin (diluted 1:200 in PBS) for 30 minutes. The muscles are rinsed for 3 x 10 minutes in PBS, followed by a 1-hour block in 2 % BSA (KPL, 50-61-00) and 0.2 % Triton X-100 (Dot Scientific Incorporated, 9002-93-1) in PBS*, and incubated overnight while rocking at 4 &deg;C in a cocktail of primary antibodies diluted in the same 2% BSA/0.2% Triton block. 
	*Glycine and blocking solutions are made on the same day as muscle harvesting and stirred for 30-60 minutes at room temperature prior to use. 
	<ol>
		<li>To mark axons and neuromuscular synapses, we use a combination of a mouse monoclonal neurofilament marker (Covance, SMI-312R; diluted 1:1000), a mouse monoclonal synaptic vesicle marker ( SV2 DSHB, diluted 1:1000), and rhodamine conjugated alpha-bungarotoxin (Sigma- Aldrich, diluted 1:200). To mark reactive Schwann cells we use rabbit anti-GAP-43 (Novus Biologicals, NB300-143, diluted 1/500). The primary antibodies are visualized with an IgG1 subtype specific fluorescein conjugated goat anti-mouse secondary antibody and DyLight 649 conjugated donkey anti-rabbit (Jackson ImmunoResearch, diluted 1:200).</li>
	</ol>
	</li>
	<li>The following day, the muscles are rinsed in PBS for 3 x 10 minutes, and incubated in secondary antibodies diluted 1:200 (in 2% BSA/0.2% Triton blocking solution). They are then rinsed 2 x 10 minutes in PBS, followed by a 5-minute immersion in DAPI (Invitrogen, D3571; diluted to 300 nM in deionized water), and another 10-minute rinse in PBS.</li>
	<li>Each muscle is then dissected on a black sylgard (Fisher Scientific, NC9492579) coated petri dish. Initially the tendons are removed at their insertion points into the muscle, and then the muscles are thinned by peeling away interior muscle fibers (Figure 3, panels A-C). Care is taken to preserve exterior surface of the muscle, including the endplate bands (Figure 3, panels D-F). The resulting muscles are mounted on plus slides (Fisher Scientific, 12-550-15) with vectashield (Vector Laboratories, H-1000) and 22 x 40 mm glass coverslips (Fisher Scientific, 12-548-5C) sealed by clear nail polish on two sides. When properly mounted on the slide the endplate band contacts the coverslip.</li>
</ol>
6. Measuring Regeneration
<ol>
	<li>Regeneration may be estimated in all three muscles (peroneus, EDL, and soleus). We often examine regeneration fourteen days after nerve crush, a time at which a substantial percentage of NMJs are re-innervated. Both earlier and later time points are also suitable, depending upon the scientific question being asked. In each muscle, re-innervation of the entire endplate band is examined. Only en face surface NMJs are scored. In this fashion, at least 200 NMJs per muscle may be quickly examined.</li>
	<li>To score muscle re-innervation we determine a "re-innervation ratio". The denominator is the number of denervated neuromuscular junctions as labeled by &alpha;-bungarotoxin binding and Schwann cell GAP-43 immunoreactivity. The numerator is the number of re-innervated NMJs as labeled by neurofilament/SV2 immunoreactivity. If needed, NMJs may be classified as either partially (incomplete coverage of &alpha;-bungarotoxin by SV2) or fully (complete coverage of &alpha;-bungarotoxin by SV2) re-innervated. For examples of re-innervated and denervated NMJs see Figure 3, panels G and H.</li>
</ol>
7. Representative Results
<img alt="Figure 1" src="/files/ftp_upload/3606/3606fig1.jpg" /> 
Figure 1. Schematic of hind limb anatomy important to nerve crush. A. A semi-circular skin incision has been made revealing the underlying musculature. B. The gluteal musculature has been separated, and the sciatic nerve revealed (step 2.4 above). An arrow denotes the approximate crush site. Retractor placement is shown as a general guide, and is adjusted during each surgery to ease visualization of the sciatic and approach of the hemostatic forceps for crush. B (inset): Placement of the sciatic on the lower jaw of the hemostatic forceps just before crush (step 2.5 above). Separate fascicles are labeled to demonstrate that they are adjacent horizontally, but not vertically, during the crush. Although three fascicles are labeled in this diagram, one may also see a fourth fascicle, the articular branch of the peroneal. For a more detailed anatomical drawing of the branching patterns of the sciatic nerve fascicles distal to the crush point, please refer to Greene's Anatomy of the Rat, Figure 18817.
<img alt="Figure 2" src="/files/ftp_upload/3606/3606fig2.jpg" /> 
Figure 2. A carbon-marked crush site in situ and toluidine blue-stained, semi-thin sections of crushed sciatic nerve. A. An example of a crush site in situ (left hind limb). Black carbon denotes the crush site. The asterisk marks a branch of the tibial nerve that innervates thigh musculature and serves as a useful landmark during crush surgery. The tibial division of the sciatic nerve is outlined in blue, the peroneal in green, and the sural in yellow. B. Semi-thin sections demonstrating a complete crush performed with hemostatic forceps. C. Semi-thin sections demonstrating an incomplete crush performed with angled forceps. In both images degenerating myelin profiles are marked with arrowheads. In panel C arrows mark examples of preserved myelin profiles and a cluster of spared axons. Scale bar is 10 &mu;m.
<img alt="Figure 3" src="/files/ftp_upload/3606/3606fig3.jpg" /> 
Figure 3. Whole mount muscle schematic and representative NMJs. A-C: Rendered images of EDL (A), peroneus longus (B), and soleus (C) muscles after removal from hind limb. Muscles shown are from right hind limb. Knee and ankle anatomical orientation are also included for reference. Tendons are colored white and outlined in a solid black line when positioned on top of the muscle. They are outlined in a black dashed line when they extend beneath the muscle. Cuts are shown in red dashed lines. Muscle is peeled away from cut sites, as indicated by blue arrows, and subsequently thinned. After thinning, the muscle is separated from the pins by cutting around what remains of the pinned tendons. D-F: Rendered images from whole mount muscles after removal of connective tissue and subsequent thinning. The endplate bands are outlined on each muscle. G-H: Soleus whole mount muscle fourteen days after sciatic nerve crush demonstrating re-innervated (G) and denervated (H) neuromuscular junctions. In these panels, NMJs, axons, and Schwann cells are visualized as described above in section 5.5.1. Acetylcholine receptors are red, axons green, and Schwann cell processes blue. In panel G, the arrows indicate areas of the NMJ that have been re-innervated by an axon. In panel H arrowheads indicate GAP-43 positive Schwann cell processes. Note the absence of axons. Scale bar is 10 &mu;m.

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	<li>Pan, Y. A., Misgeld, T., Lichtman, J. W., Sanes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+neurotoxic+and+neuroprotective+agents+on+peripheral+nerve+regeneration+assayed+by+time-lapse+imaging+in+vivo.">Effects of neurotoxic and neuroprotective agents on peripheral nerve regeneration assayed by time-lapse imaging in vivo.</a> J. Neurosci. 23, 11479-11488 (2003).</li><li>Magill, C., Tong, A., Kawamura, D., Hayashi, A., Hunter, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reinnervation+of+the+tibialis+anterior+following+sciatic+nerve+crush+injury:+A+confocal+microscopic+study+in+transgenic+mice.">Reinnervation of the tibialis anterior following sciatic nerve crush injury: A confocal microscopic study in transgenic mice.</a> Exp. Neurol. 207, 64-74 (2007).</li><li>Amado, S., Sim&#241;es, M. J., Armada-da-Silva, P. A. S., Lu&#237;s, A. L., Shirosaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+hybrid+chitosan+membranes+and+N1E-115+cells+for+promoting+nerve+regeneration+in+an+axonotmesis+rat+model.">Use of hybrid chitosan membranes and N1E-115 cells for promoting nerve regeneration in an axonotmesis rat model.</a> Biomaterials. 29, 4409-4419 (2008).</li><li>Lu&#237;s, A. L., Rodrigues, J. M., Geuna, S., Amado, S., Sim&#240;es, M. 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We have nothing to disclose.

We have presented a method to obtain a reliably complete sciatic nerve crush with precise marking of the crush site. As previously mentioned, sciatic nerve crush is a common model of peripheral nerve injury in mice and rats. Although each method of crush has its advantages and disadvantages, we found this method produced a complete crush that was easily marked with a minimum of special equipment (e.g. special clamps, etc).
Crush Methods
The most common instrument use...

<table border="1">
<tbody>
 <tr>
 <td>Name (description/quantity)</td>
 <td>Supplier</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>Mini Clipper with No. 0000 Blade</td>
 <td>Roboz</td>
 <td>RC-5903</td>
 </tr>
 <tr>
 <td>Nair Hair Remover (9 oz.)</td>
 <td>Church and Dwight Co., Inc.</td>
 <td>N/A</td>
 </tr>
 <tr>
 <td>Betadine Surgical Scrub (1 gallon)</td>
 <td>Fisher Scientific</td>
 <td>19066452</td>
 </tr>
 <tr>
 <td>Ophthalmic Ointment (1 oz.)</td>
 <td>Fisher Scientific</td>
 <td>19082795</td>
 </tr>
 </tbody>
</table>
Surgical Tools:
<table border="1">
<tbody>
 <tr>
 <td>Name (description/quantity)</td>
 <td>Supplier</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>FST 250 Hot bead sterilizer</td>
 <td>Fine Science Tools</td>
 <td>18000-45</td>
 </tr>
 <tr>
 <td>Iris Scissors (11 cm long)</td>
 <td>World Precision Instruments</td>
 <td>500216</td>
 </tr>
 <tr>
 <td>Potts-Smith Forceps (Straight; 18cm; 1x2 Teeth)</td>
 <td>Fine Science Tools</td>
 <td>11024-18</td>
 </tr>
 <tr>
 <td>McPherson-Vannas Scissors (5 mm blades) </td>
 <td>World Precision Instruments</td>
 <td>14124-G</td>
 </tr>
 <tr>
 <td>Dumont #5 Forceps &#x2013; Dumoxel Standard Tip</td>
 <td>Fine Science Tools</td>
 <td>11252-30</td>
 </tr>
 <tr>
 <td>Dumont #5/45 Forceps &#x2013; Dumoxel Standard Tip</td>
 <td>Fine Science Tools</td>
 <td>11251-35</td>
 </tr>
 <tr>
 <td>Ultra Fine Hemostats (Straight; Smooth Inside Surfaces) </td>
 <td>Fine Science Tools</td>
 <td>13020-12</td>
 </tr>
 <tr>
 <td>Powdered Activated Carbon (500 g)</td>
 <td>Fisher Scientific</td>
 <td>C272-500</td>
 </tr>
 <tr>
 <td>Size 6-0 Sutures with C-1 Needle (Sterile, Silk, Black, Braided , Non-absorbable; 18&#x201D; Length; Box of 36)</td>
 <td>Roboz</td>
 <td>SUT-1073-11</td>
 </tr>
 <tr>
 <td>Reflex Clip Applier (for 9 mm clips) </td>
 <td>World Precision Instruments</td>
 <td>500345</td>
 </tr>
 <tr>
 <td>9mm Stainless Steel Reflex Clips (100/box) </td>
 <td>World Precision Instruments</td>
 <td>500346</td>
 </tr>
 </tbody>
</table>

Animal Care:
<table border="1">
<tbody>
 <tr>
 <td>Name (description/quantity)</td>
 <td>Supplier</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>0.9% Sodium Chloride Injection (Preservative Free; 20 mL)</td>
 <td>Hospira</td>
 <td>0409-4888-20</td>
 </tr>
 <tr>
 <td>Complete Homeothermic Blanket System with Flexible Probe (Medium, 115 VAC, 60 Hz)</td>
 <td>Harvard Apparatus</td>
 <td>507222F</td>
 </tr>
 </tbody>
</table>
Surgical Platform and Retractors:
 (purchased at local hardware store)
<table border="1">
<tbody>
 <tr>
 <td>Name (description/)</td>
 <td>Quantity</td>
 </tr>
 <tr>
 <td>Stainless Steel Platform (Milled; ~12&#x201D;x12&#x201D;x1/8&#x201D;)</td>
 <td>1</td>
 </tr>
 <tr>
 <td>Button Magnets (Example: Eclipse E825)</td>
 <td>3</td>
 </tr>
 <tr>
 <td>Stainless Steel Bolts (3&#x201D; Long; Diameter Determined by Button Magnets)</td>
 <td>3</td>
 </tr>
 <tr>
 <td>Stainless Steel Nuts (Sized to Fit Bolts)</td>
 <td>9</td>
 </tr>
 <tr>
 <td>Rubber Bands (Light Tension)</td>
 <td>3</td>
 </tr>
 <tr>
 <td>Insect Pins (Both Ends Bent to Form Hooks)</td>
 <td>3</td>
 </tr>
 </tbody>
</table>
Semi-Thins:
<table border="1">
<tbody>
 <tr>
 <td>Name (description/quantity)</td>
 <td>Supplier</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>Paraformaldehyde (1 kg)</td>
 <td>Sigma-Aldrich</td>
 <td>P6148-1KG</td>
 </tr>
 <tr>
 <td>Sodium Phosphate Dibasic Anhydrous (500 g; Used to Prepare Phosphate Buffer)</td>
 <td>Fisher Scientific</td>
 <td>S375-500 </td>
 </tr>
 <tr>
 <td>Sodium Phosphate Monobasic Anhydrous (1 kg; Used to Prepare Phosphate Buffer)</td>
 <td>Fisher Scientific</td>
 <td>AC38987-0010</td>
 </tr>
 <tr>
 <td>Glutaraldehyde (50%; 10 x 10 mL)</td>
 <td>Ted Pella, Inc.</td>
 <td>18431</td>
 </tr>
 <tr>
 <td>Osmium Tetroxide (4% Aqueous, 10 x 10 mL)</td>
 <td>Ted Pella, Inc.</td>
 <td>18465</td>
 </tr>
 <tr>
 <td>Propylene Oxide (450 mL)</td>
 <td>Ted Pella, Inc.</td>
 <td>18601</td>
 </tr>
 <tr>
 <td>Embed 812 (Kit, for hard blocks/high image contrast)</td>
 <td>Electron Microscopy Sciences</td>
 <td>14120</td>
 </tr>
 <tr>
 <td>Toluidine Blue (25 g)</td>
 <td>Ted Pella, Inc.</td>
 <td>19451</td>
 </tr>
 </tbody>
</table>
Whole Mount Muscle Preparation and Immunohistochemistry:
<table border="1">
<tbody>
 <tr>
 <td>Name (description/quantity)</td>
 <td>Supplier</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>Paraformaldehyde (1 kg)</td>
 <td>Sigma-Aldrich</td>
 <td>P6148-1KG</td>
 </tr>
 <tr>
 <td>Sodium Phosphate Dibasic Anhydrous (500 g; Used to Prepare Phosphate Buffer)</td>
 <td>Fisher Scientific</td>
 <td>S375-500 </td>
 </tr>
 <tr>
 <td>Sodium Phosphate Monobasic Anhydrous (1 kg; Used to Prepare Phosphate Buffer)</td>
 <td>Fisher Scientific</td>
 <td>AC38987-0010</td>
 </tr>
 <tr>
 <td>10% BSA Diluent/Blocking Solution (200 mL)
 </td>
 <td>Kirkegaard &amp; Perry Laboratories, Inc.</td>
 <td>50-61-00</td>
 </tr>
 <tr>
 <td>Triton X-100 (100 mL)</td>
 <td>Dot Scientific Incorporated</td>
 <td>9002-93-1</td>
 </tr>
 <tr>
 <td>Glycine, 98% (1 kg)</td>
 <td>Fisher Scientific</td>
 <td>AC12007-0010</td>
 </tr>
 <tr>
 <td>Tissue-Tek Cryo-OCT Compound (Case of 12; 4 oz bottles)</td>
 <td>Fisher Scientific</td>
 <td>14-373-65</td>
 </tr>
 <tr>
 <td>Sylgard DOW 170 (2 lb. Kit)</td>
 <td>Fisher Scientific</td>
 <td>NC9492579</td>
 </tr>
 <tr>
 <td>Stainless Steel Insect Pins, Size 1 (100/pkg)</td>
 <td>Fine Science Tools</td>
 <td>26001-40</td>
 </tr>
 <tr>
 <td>Tetramethylrhodamine-A-Bungarotoxin (0.5 mg)</td>
 <td>Sigma-Aldrich</td>
 <td>T0195-.5MG</td>
 </tr>
 <tr>
 <td>Mouse Monoclonal Antibody Against SMI-312 (0.1 mL)</td>
 <td>Covance</td>
 <td>SMI-312R</td>
 </tr>
 <tr>
 <td>Mouse Monoclonal Antibody Against SV2 (0.1 mL)</td>
 <td>Developmental Studies Hybridoma Bank (DSHB)</td>
 <td>SV2</td>
 </tr>
 <tr>
 <td>Rabbit Polyclonal Antibody Against GAP-43</td>
 <td>Novus Biologicals</td>
 <td>NB300-143</td>
 </tr>
 <tr>
 <td>Flourescein Conjugated Goat Anti-Mouse IgG, Fc&#x03B3;&nbsp;Subclass 1 Specific</td>
 <td>Jackson ImmunoResearch</td>
 <td><a href="http://www.jacksonimmuno.com/MERCHANT2/merchant.mv?Screen=BASK&amp;Store_Code=JI&amp;Action=ADPR&amp;Product_Code=115-095-205&amp;Attributes=Yes&amp;Quantity=1">115-095-205</a></td>
 </tr>
 <tr>
 <td>DyLight 649 Conjugated Donkey Anti-Rabbit IgG</td>
 <td>Jackson ImmunoResearch</td>
 <td><a href="http://www.jacksonimmuno.com/MERCHANT2/merchant.mv?Screen=BASK&amp;Store_Code=JI&amp;Action=ADPR&amp;Product_Code=711-495-152&amp;Attributes=Yes&amp;Quantity=1">711-495-152</a></td>
 </tr>
 <tr>
 <td>4',6-diamidino-2-phenylindole (DAPI, dilactate; 10 mg)</td>
 <td>Invitrogen</td>
 <td>D3571</td>
 </tr>
 <tr>
 <td>Vectashield Mounting Medium (10 mL)</td>
 <td>Vector Laboratories</td>
 <td>H-1000</td>
 </tr>
 <tr>
 <td>Superfrost Plus Microscope Slides (White; Size: 75 x 25 mm; Pack of 144)</td>
 <td>Fisher Scientific</td>
 <td>12-550-15</td>
 </tr>
 <tr>
 <td>Fisherfinest Premium Cover Glasses (Size: 40 x 22 mm; Pack of 1 oz.)</td>
 <td>Fisher Scientific</td>
 <td>12-548-5C</td>
 </tr>
 </tbody>
</table>

reproducible mouse sciatic nerve crush and subsequent assessment of regeneration by whole mount muscle analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

Watch this Scientific Journal Video about Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis at JoVE.com

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

reproducible-mouse-sciatic-nerve-crush-subsequent-assessment

Research

JoVE Journal

Neuroscience

33.5K Views.  Temple University. In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Video: Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Axoplasm Isolation from Rat Sciatic Nerve

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we demonstrate and describe a protocol for axoplasm isolation from adult rat sciatic nerve based on the following steps: (1) dissection of nerve fascicles and separation of connective tissue; (2) incubation of short segments of nerve fascicles in hypotonic medium to release myelin and lyse non-axonal structures; and (3) extraction of the remaining axon-enriched material. Proteomic and biochemical characterization of this preparation has confirmed a high degree of enrichment for axonal components.

We demonstrate a protocol for axoplasm isolation from adult rat sciatic nerve based on dissection of nerve fascicles and incubation in hypotonic medium to release myelin and lyse non-axonal structures, followed by extraction of the remaining axon-enriched material.

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla.

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells ...

Multifactorial Assessment of Motor Behavior in Rats after Unilateral Sciatic Nerve Crush Injury

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In addition, in the research field of movement disorders, sciatic crush injury has been employed to trigger a dystonia-like phenotype in genetically predisposed DYT-TOR1A rodent models of dystonia. To achieve consistent, reproducible and comparable results after a sciatic nerve crush injury, a standardized method for inducing the nerve crush is essential, in addition to a standardized phenotypical characterization. Attention must be paid not only to the specific assortment of behavioral tests, but also to the technical requirements, the correct execution and consecutive data analysis. This protocol describes in detail how to perform a sciatic nerve crush injury and provides a behavioral test battery for the assessment of motor deficits in rats that includes the open field test, the CatWalk XT gait analysis, the beam walking task, and the ladder rung walking task.

We provide a protocol for the assessment of motor behavior via a behavioral test battery in rats after sciatic nerve crush injury.

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In ...

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...

Methods Article

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis