This work was funded in part by NIH R01 #DE021475 to CTM and an NSF Graduate Research Fellowship to NAE. Thanks to Miles Johnson for assistance with imaging and Priscilla Erickson for critical reading of the manuscript.

All fish work was approved by the Institutional Animal Care and Use Committee of the University of California-Berkeley (protocol number R330). Euthanasia was performed using immersion in 0.025% Tricaine-S buffered with 0.1% sodium bicarbonate39. All steps are performed at room temperature.
1. Preparation&#160;
Note: Perform steps 1.1-1.5 in conical tubes or scintillation vials that can seal tightly and be laid horizontally. Fish do not need to be constantly shaken, but try to mix the solution as often as possible by gently inverting or shaking the rack of tubes or vials to expose all sides of the fish to the staining solution and allow stain to penetrate the tissue evenly. Do not place a large batch of fish on a platform shaker, as the heavy weight of the liquid will break the shaker.
<ol>
	<li>Fix either freshly euthanized fish or fish stored in ethanol with 10% Neutral Buffered Formalin (NBF) overnight. Alternatively, use 4% paraformaldehyde in 1x PBS solution instead of 10% NBF. 
	Note: If extracting DNA, clip a small portion of the caudal or pectoral fins prior to fixation and store in ethanol.</li>
	<li>Dispose of fix properly in a chemical hood and replace with tap water (that is ~pH 7.0) for 2 hr. Avoid using de-ionized water as it can often be acidic and can decalcify bone.</li>
	<li>Remove water and stain fish with 0.008% Alizarin Red S in 1% KOH in water for 24 hr. For fish less than 20 mm standard length, use 0.004% Alizarin Red S. (Make a 100x (0.8%) stock solution of Alizarin Red S which can then be diluted).</li>
	<li>Remove stain (putting in appropriate waste container in hood) and place fish in tap water for a few hr. Change water as needed until water rinse is mostly clear.</li>
	<li>Remove water and place fish into 50% glycerol, 0.25% KOH for mild clearing and subsequent dissection. 
	Note: This staining protocol is modified from previously described methods40,41.</li>
</ol>
2. Dissection&#160;
Note: See Figure 1 for a review of relevant head skeletal morphology.
<img alt="Figure 1" src="/files/ftp_upload/54056/54056fig1.jpg" /> 
Figure 1: Stickleback head skeletal morphology. Alizarin Red stained threespine stickleback head imaged with fluorescence under a rhodamine B filter set. Useful morphology is labeled: Op = opercle, Subop = subopercle, BSRs = branchiostegal rays, Preop = preopercle, Infraorb 1-3 = infraorbital 1-3 (also called circumorbitals or suborbitals), Dent = dentary, Premax = premaxilla, Max = maxilla, Nas = nasal, Lat. ethm = lateral ethmoid, Psph = parasphenoid, Fron = frontal bone. For a more detailed description of the stickleback head skeleton, see Anker (1974)31. <a href="https://www.jove.com/files/ftp_upload/54056/54056fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Lay the fish flat (Figure 2A) and insert sharp #5 watchmaker&#39;s forceps into the side of the eye at a ~45&#176; angle to puncture the membrane covering the eye.</li>
	<li>Peel the membrane away from the eye, similar to peeling a yogurt lid (Figure 2B).</li>
	<li>Insert open forceps behind the eye, grab hold of the optic nerve behind the eye, and remove the eye (Figure 2C). Do not puncture the eye as it will leak melanin. If punctured, melanin can be washed away during later steps.</li>
	<li>Repeat on other side.</li>
	<li>Starting from the posterior, place one small dissecting scissor blade under the opercle flap, drag scissor blade dorsally above opercle, then cut soft tissue through to the eye socket (Figure 2D). Cut dorsal to the opercle bone.</li>
	<li>Cut the frontal bone (dorsal to the eye socket) (Figure 2E).</li>
	<li>Cut the midline parasphenoid bone around the center of the eye sockets (Figure 2F).</li>
	<li>Repeat opercle cut on the opposing side.</li>
	<li>Insert forceps under the opercle and slowly peel the face away from the body, trimming any soft tissue still attached (Figure 2G-H). Take care not to disrupt the first row of gill rakers.
	<ol>
		<li>With forceps, detach the ceratohyals on both sides from the midline basihyal while peeling away and removing the anterior craniofacial skeleton (the entire jaw including the dentary, premaxilla, and maxilla; the entire hyoid skeleton including the exterior dermal opercle, preopercle, subopercle, and branchiostegal rays and the underlying dorsal and ventral endochondral elements; and the anterior part of the skull including the nasal, lateral ethmoid, and infraorbital bones, see Figures 1 and 2I).</li>
		<li>Pelvic spines can be folded out from the body, and can serve as a handle for forceps to grab hold of when present. Spines lock into place. To unlock, gently pull spine with forceps directly away from fish body, then gently bend posteriorly to press spine flat against fish.</li>
	</ol>
	</li>
	<li>Insert closed forceps posterior and ventral to the branchial skeleton (just below gut tube) and drag forceps anteriorly, teasing apart the remaining muscles and ligaments attached to the branchial skeleton (Figure 2J-K).</li>
	<li>Using the tips of closed forceps, scrape away the muscles attaching the dorsal branchial skeleton to the ventral braincase in a posterior to anterior direction (Figure 2L).</li>
	<li>Repeat 2.9 and 2.10 on the opposing side.</li>
	<li>Grasp the base of the gut tube and pull anteriorly to remove the branchial skeleton and gut tube (Figure 2M-N).</li>
	<li>Separate the gut tube by making a perpendicular cut posterior to the end of the fifth ceratobranchial (Figure 2O).</li>
	<li>After removing any remaining bone fragments from the braincase on the dorsal side of the branchial skeleton, insert scissors into the branchial basket to make a dorsal cut (cutting anterior to posterior) between the bilateral sets of dorsal tooth plates (Figure 3A-D). Ensure cut is centered to avoid damaging the dorsal tooth plates.</li>
	<li>Make two shallow lateral cuts in the rubbery gut lumen at the posterior end of the branchial skeleton (anterior end of gut tube) to assist with opening the branchial skeleton (Figure 3E).</li>
	<li>Place fish and all tissue pieces into a jar and place the branchial skeleton into a microcentrifuge tube with 50% glycerol, 0.25% KOH to continue gentle clearing, or 100% glycerol if no further clearing is required. Label jars and tubes with a unique identifier so they can be tracked. The amount of clearing required is largely a function of the size of the fish, large adult fish (over 40 mm in standard length) typically require additional clearing.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/54056/54056fig2.jpg" /> 
Figure 2: Stickleback branchial skeleton dissection. Alizarin Red stained threespine stickleback fish ready for dissection. The eye is depigmented from extensive clearing. Blue arrows indicate the direction of motion. (A) Lateral view of stickleback head, anterior is to the right. (B) Removal of membrane covering the eye. (C) Removal of the eye. (D) Dorsal cut above the opercle. (E) Frontal bone cut. (F) Parasphenoid cut. (G-I) Removal of the facial skeleton. (J) Removal of ventral branchial skeleton soft tissue connections. (K-L) Removal of dorsal branchial skeleton connections. (M-N) Removal of the branchial skeleton. (O) Separate the gut tube from the branchial skeleton. See steps 2.1 through 2.16 for more details. Scale bar = 5 mm. <a href="https://www.jove.com/files/ftp_upload/54056/54056fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Branchial Skeleton Re-staining (If Necessary)
<ol>
	<li>To stain the branchial skeleton darker or clear tissue more, remove 50% glycerol, 0.25% KOH solution and wash with 1% KOH twice (one five minute wash followed by a second 24 hr wash while shaking horizontally on a platform shaker).</li>
	<li>Remove 1% KOH and re-stain with 0.008% Alizarin Red S in 1% KOH for 24 hr.</li>
	<li>Remove stain and replace with 1% KOH for 24 hr.</li>
	<li>Remove KOH solution and replace with 50% glycerol, 0.25% KOH.</li>
</ol>
4. Mounting Branchial Skeleton
<ol>
	<li>Remove branchial skeleton from 50% glycerol, 0.25% KOH or 100% glycerol and place near the bottom of a 22 mm x 60 mm glass cover slip with the dorsal side facing up (Figure 3F). Add a few drops of 50% glycerol, 0.25% KOH or 100% glycerol on top of branchial skeleton. If transitioning from 50% glycerol, 0.25% KOH to 100% glycerol, change solution in a microcentrifuge tube and shake for &#62;5 min prior to mounting to equilibrate tissue.</li>
	<li>Roll out two small balls of modeling clay and place on either end of the cover slip to act as spacers.</li>
	<li>Loosely place a second coverslip on top with enough pressure to flatten the anterior branchial skeleton (Figure 3G).</li>
	<li>Peel open the left dorsal flap including dorsal tooth plates, flatten, and slide between the coverslips (Figure 3H).</li>
	<li>Repeat technique with right dorsal flap and push entire branchial skeleton away from the edge of the coverslip (Figure 3I).
	<ol>
		<li>Alternatively, hold both dorsal flaps open with forceps and carefully place the coverslip on, flattening the branchial skeleton in one smooth motion.</li>
		<li>Alternatively, mount the branchial skeleton upside-down on one cover slip, splaying each dorsal side out laterally so gravity does not allow the branchial skeleton to close back up. Then cover with second 22 mm x 60 mm glass cover slip and invert prep. 
		Note: Different mounting techniques tend to work better or worse for each individual. Try each and see what feels most comfortable.</li>
	</ol>
	</li>
	<li>Lightly press on top coverslip to flatten clay balls enough to keep the branchial skeleton mounted flat, but take care not to crush the specimen.
	<ol>
		<li>During the mounting process, ceratobranchials may rotate and obscure a row of rakers. Remedy this by sliding forceps between the coverslips and re-orienting the ceratobranchials or entire branchial skeleton.</li>
	</ol>
	</li>
	<li>Store preps flat in slide trays at room temperature. Mounted in 100% glycerol, preps can be stored between bridged coverslips for at least a decade. Clean forceps and scissors with isopropanol or ethanol and cover tips.</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/54056/54056fig3.jpg" /> 
Figure 3: Flat mounting the branchial skeleton. Manipulation and mounting of the branchial skeleton is shown. Blue arrows indicate the direction of motion. (A) Branchial skeleton dorsal side up. (B-D) Rotation and incision between the dorsal tooth plates. (E) Lateral cut in soft tissue to further open the base of the gut tube. (F) Branchial skeleton placed at the bottom of a coverslip ready for mounting. (G) Second coverslip placed on the anterior half of the branchial skeleton (above dorsal tooth plates). (H-I) Flat mounting of the branchial skeleton by opening dorsal tooth plate flaps and sliding between two cover slips. See steps 4.1 through 4.6 for more details. Scale bar = 5 mm. <a href="https://www.jove.com/files/ftp_upload/54056/54056fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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=Evolved+tooth+gain+in+sticklebacks+is+associated+with+a+cis-regulatory+allele+of+Bmp6.">Evolved tooth gain in sticklebacks is associated with a cis-regulatory allele of Bmp6.</a> Proc. Natl. Acad. Sci. 111 (38), 13912-13917 (2014).</li><li>Ellis, N. 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=Distinct+developmental+and+genetic+mechanisms+underlie+convergently+evolved+tooth+gain+in+sticklebacks.">Distinct developmental and genetic mechanisms underlie convergently evolved tooth gain in sticklebacks.</a> Development. (142), 2442-2451 (2015).</li><li>Tucker, A. S., Fraser, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+and+developmental+diversity+of+tooth+regeneration.">Evolution and developmental diversity of tooth regeneration.</a> Semin. Cell Dev. Biol. 25-26, 71-80 (2014).</li><li>Erickson, P. A., Glazer, A. M., Cleves, P. A., Smith, A. S., Miller, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+developmentally+temporal+quantitative+trait+loci+underlie+convergent+evolution+of+increased+branchial+bone+length+in+sticklebacks.">Two developmentally temporal quantitative trait loci underlie convergent evolution of increased branchial bone length in sticklebacks.</a> Proc. R. Soc. B. 281, (2014).</li><li>Leary, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> AVMA Guidelines for the Euthanasia of Animals. , (2013).</li><li>Bell, 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=Evolutionary+phenetics+and+genetics.">Evolutionary phenetics and genetics.</a> Evol. Genet. Fishes. , 431-528 (1984).</li><li>Taylor, W. R., Van Dyke, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revised+procedures+for+staining+and+clearing+small+fishes+and+other+vertebrates+for+bone+and+cartilage+study.">Revised procedures for staining and clearing small fishes and other vertebrates for bone and cartilage study.</a> Cybium. 9 (2), 107-119 (1985).</li><li>Erickson, P. 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=A+190+base+pair,+TGF-&#946;+responsive+tooth+and+fin+enhancer+is+required+for+stickleback+Bmp6+expression.">A 190 base pair, TGF-&#946; responsive tooth and fin enhancer is required for stickleback Bmp6 expression.</a> Dev. Biol. 401 (2), 310-323 (2015).</li><li>Miller, C. T., 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=cis-Regulatory+changes+in+Kit+ligand+expression+and+parallel+evolution+of+pigmentation+in+sticklebacks+and+humans.">cis-Regulatory changes in Kit ligand expression and parallel evolution of pigmentation in sticklebacks and humans.</a> Cell. 131 (6), 1179-1189 (2007).</li><li>Aigler, S. R., Jandzik, D., Hatta, K., Uesugi, K., Stock, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+and+constraint+underlie+irreversibility+of+tooth+loss+in+cypriniform+fishes.">Selection and constraint underlie irreversibility of tooth loss in cypriniform fishes.</a> Proc. Natl. Acad. Sci. U. S. A. 111 (21), 7707-7712 (2014).</li><li>Pasco-Viel, E., 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=Evolutionary+trends+of+the+pharyngeal+dentition+in+Cypriniformes+(Actinopterygii+Ostariophysi).">Evolutionary trends of the pharyngeal dentition in Cypriniformes (Actinopterygii Ostariophysi).</a> PLoS One. 5 (6), e11293 (2010).</li></ol>

The authors have nothing to disclose.

The branchial skeleton is a complex set of bones in the throat of a fish that manipulates, filters, and masticates food items on their way to the esophagus. Many interesting trophic traits including the patterning of gill rakers, pharyngeal teeth, and branchial bones vary across and within species. The majority of these traits are difficult to near impossible to accurately measure with the branchial skeleton in situ (e.g., gill raker length, branchial bone length). This flat-mounting protocol places all...

An incredible amount of diversity exists in the head skeleton among vertebrates, especially among fishes. In many cases this diversity facilitates different feeding strategies1-4, and can involve major changes to both external and internal craniofacial patterning. The branchial skeleton is located internally in the throat of a fish and surrounds most of the buccal cavity. The branchial skeleton is comprised of 5 serially homologous segments, the anterior four of which support the gills. Together these five segments function as an interface between fish and their food5. Variation in a multitude of traits including gill rakers, pharyngeal teeth, and branchial bones contribute to efficient foraging on different types of food.
Sticklebacks have undergone an adaptive radiation after ancestral oceanic forms colonized freshwater lakes and creeks throughout the northern hemisphere. The shift in diet from small zooplankton in the ocean to larger prey in freshwater has resulted in dramatic trophic variation in several craniofacial traits6. While many studies have focused on external craniofacial differences in sticklebacks7-13, important craniofacial changes evolve repeatedly in the internal branchial skeleton. The ability to create fertile hybrids between morphologically distinct stickleback populations provides an excellent opportunity to map the genetic basis of evolved changes to the branchial skeleton.
One trophic trait of ecological significance is the patterning of gill rakers, periodic dermal bones that line the anterior and posterior faces of the branchial bones and are used to filter prey items. Fish that typically feed on small prey items tend to have longer and more densely spaced gill rakers compared to fish that feed on larger prey14,15. Variation in gill rakers has been reported both within and between species14-19, and aspects of gill raker patterning contribute to trophic niches and fitness16. Decades of research have extensively documented gill raker number and length variation in threespine sticklebacks17-21; however, these studies typically focus on the first row of gill rakers. Recent work has shown modularity in the genetic control of gill raker number across the branchial skeleton22,23 and across a single row in gill raker spacing23 and length24 highlighting the importance of studying more than row one or a single gill raker to understand the developmental genetic basis of gill raker reduction.
A second trophic trait of both ecological and biomedical significance is the patterning of pharyngeal teeth. Teeth in fishes can be located in both the oral jaw and in the branchial skeleton, known as pharyngeal teeth. Oral teeth are used primarily for prey capture while pharyngeal teeth are used for mastication and prey manipulation25-27. Both sets form via shared developmental mechanisms and are considered developmentally homologous28. Interesting modularity occurs whereby some species, such as zebrafish, lack oral and dorsal pharyngeal teeth29 while other species have multiple toothed ceratobranchials, pharyngobranchials, and sometimes toothed basihyal and hypobranchials30. In sticklebacks, pharyngeal teeth are found ventrally on the fifth ceratobranchial and dorsally on the anterior and posterior pharyngobranchials31. Kinematics on stickleback feeding show the oral jaw is used primarily for prey capture and facilitating suction feeding9 leaving mastication to the pharyngeal jaw. In cichlids, lower pharyngeal jaw morphology varies dramatically32,33 and has been shown to be adaptive and correlated with trophic niche34. Multiple freshwater stickleback populations have evolved dramatic increases in ventral pharyngeal tooth number23,35,36. Recent work has demonstrated that the developmental genetic basis of this evolved tooth gain is largely distinct in two independently derived populations of freshwater sticklebacks36. Unlike mammalian teeth, fish regenerate their teeth continuously throughout adult life37. Both of these previously described high toothed freshwater populations have evolved an accelerated tooth replacement rate, providing a rare vertebrate system to study the genetic basis of regeneration36.
A third trophic trait that has evolved repeatedly in freshwater sticklebacks is longer epibranchial and ceratobranchial bones, the branchial arch segmental homologs of the upper and lower jaw, respectively38. Longer branchial bones confer a larger buccal cavity and likely are adaptive for allowing larger prey items to be consumed. Furthermore, in other fish, epibranchial bones are important for depression of the dorsal pharyngeal tooth plates25. Like gill rakers and pharyngeal teeth, the branchial bones are internal and thus, difficult to easily visualize or quantify.
Here we present a detailed protocol to dissect and flat-mount the branchial skeleton, allowing easy visualization and quantification of a variety of important craniofacial traits. While this protocol describes a stickleback dissection, this same method works on a variety of other fishes.

<table>
	<tbody>
		<tr>
			<td>Sodium Hydroxide (KOH)</td>
			<td>EMD</td>
			<td>PX1480-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Alderich</td>
			<td>G7893-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Neutral Buffered Formalin (NBF)</td>
			<td>Azer Scientific</td>
			<td>NBF-4-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Alizarin Red S</td>
			<td>EMD</td>
			<td>116-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Cover Glasses 22x60mm</td>
			<td>VWR</td>
			<td>16004-350</td>
			<td></td>
		</tr>
		<tr>
			<td>100x10mm Glass Petri Dish</td>
			<td>Kimble Chase</td>
			<td>23064-10010</td>
			<td>To dissect samples on</td>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Ellsworth Adhesives</td>
			<td>184 SIL ELAST KIT 0.5KG</td>
			<td>Can be poured into glass or plastic petri dishes to make dissecting plates</td>
		</tr>
		<tr>
			<td>Modeling Clay</td>
			<td>Sargent Art</td>
			<td>22-4000</td>
			<td>1lb cream</td>
		</tr>
		<tr>
			<td>Scintillation Vials (case of 500)</td>
			<td>Wheaton</td>
			<td>66021-668</td>
			<td>Borosilicate Glass with Screw Cap</td>
		</tr>
		<tr>
			<td>Forceps-Dumont #5 Inox (Biologie tip)</td>
			<td>FST</td>
			<td>11252-20</td>
			<td>Dumostars are an alternative</td>
		</tr>
		<tr>
			<td>Dissecting Scissors&#160;</td>
			<td>FST</td>
			<td>15003-08</td>
			<td>Alternate sizes are available depending on size of sample</td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Leica</td>
			<td>S6E with KL300 LED</td>
			<td>Many other models work nicely, having a flat base helps</td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes 1.7mL</td>
			<td>Denville</td>
			<td>C-2170</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardboard slide tray</td>
			<td>Fisher</td>
			<td>12-587-10</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection and flat-mounting of the threespine stickleback branchial skeleton

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology of the fish branchial skeleton is matched to a species' diet. Threespine stickleback fish (Gasterosteus aculeatus) have emerged as a model system to study the genetic and developmental basis of evolved differences in a variety of traits. Marine populations of sticklebacks have repeatedly colonized countless new freshwater lakes and creeks. Adaptation to the new diet in these freshwater environments likely underlies a series of craniofacial changes that have evolved repeatedly in independently derived freshwater populations. These include three major patterning changes to the branchial skeleton: reductions in the number and length of gill raker bones, increases in pharyngeal tooth number, and increased branchial bone lengths. Here we describe a detailed protocol to dissect and flat-mount the internal branchial skeleton in threespine stickleback fish. Dissection of the entire three-dimensional branchial skeleton and mounting it flat into a largely two-dimensional prep allows for the easy visualization and quantification of branchial skeleton morphology. This dissection method is inexpensive, fast, relatively easy, and applicable to a wide variety of fish species. In sticklebacks, this efficient method allows the quantification of skeletal morphology in genetic crosses to map genomic regions controlling craniofacial patterning.

This protocol results in a dissected and flat mounted branchial skeleton (Figure 4) where a variety of important trophic traits can be quantified. From a dorsal view, all rows of gill rakers, all pharyngeal tooth plates, and nearly all branchial bones can be easily visualized and quantified22-24,35,36,38,42. Alizarin Red S also fluoresces on a rhodamine or similar red filter allowing double labeling with other markers (e.g., trans...

Watch this Scientific Journal Video about Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton at JoVE.com

Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton

The branchial skeleton, including gill rakers, pharyngeal teeth, and branchial bones, serves as the primary site of food processing in most fish. Here we describe a protocol to dissect and flat-mount this internal skeleton in threespine sticklebacks. This method is also applicable to a variety of other fish species.

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology ...

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9.7K Views.  University of California, Berkeley. The branchial skeleton, including gill rakers, pharyngeal teeth, and branchial bones, serves as the primary site of food processing in most fish. Here we describe a protocol to dissect and flat-mount this internal skeleton in threespine sticklebacks. This method is also applicable to a variety of other fish species. 

Video: Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton

Dissection of Drosophila Ovaries

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of the stomach is defined by the mouth opening and the posterior end by the bottom of the pylorus. Along the dorsal side of the stomach lies the stomatogastric nervous system (STNS). This nervous system, which contains rhythmic networks that underlie feeding behavior, is an established model system for studying rhythm generating networks and neuromodulation 1,2. While it is possible to study this system in vivo 3, the STNS continues to produce its rhythmic activity when isolated in vitro. In order to study this system in vitro the stomach must be removed from the animal. This video article describes how the stomach can be dissected from the American lobster. In an accompanying video article4 we demonstrate how the STNS can be isolated from the stomach.

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

We describe the gross dissection of the stomach of the American lobster (Homarus americanus).

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...

Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with conspecifics at a close range. These displays are achieved by contraction of the mimetic muscles, which are skeletal muscle attached to the dermis of the face. Reverse dissection, removing the full facial mask from the skull and approaching mimetic muscles in reverse, is an effective but destructive way of revealing the morphology of mimetic muscles but it is destructive. DiceCT is a novel mechanism for visualizing skeletal muscles, including mimetic muscles, and isolating individual muscle fascicles for quantitative measurement. Additionally, DiceCT provides a non-destructive mechanism for visualizing muscles. The combined techniques of reverse dissection and DiceCT can be used to assess the evolutionary morphology of mimetic musculature as well as potential contraction strength and velocity in these muscles. This study further demonstrates that DiceCT can be used to accurately and reliably visualize mimetic muscles as well as reverse dissection and provide a non-destructive method for sampling mimetic muscles.

Facial expressions are a mode of visual communication produced by mimetic muscles. Here, we present protocols for the novel techniques of reverse dissection and DiceCT to fully visualize and assess mimetic muscles. These combined techniques can examine both morphological and physiological aspects of mimetic musculature to determine functional aspects.

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with ...

Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton

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Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face