The authors thank Masaki Yoda and Elise Lamar for critical reading of the manuscript, Kazumasa Takenouchi for help with histology, Mari Fujiwara for help with microscopy and Makoto Morikawa for help in photographing human and mouse auditory ossicles.

All animal procedures performed in this study are approved by the Keio University Institutional Animal Care and Use Committee (IACUC - approval number: 09221) and follow the Institutional Guidelines on Animal Experimentation at Keio University for the use of animals in research. Human specimens were isolated from a cadaver donated to the Department of Anatomy, Keio University School of Medicine, and were used in accordance with institutional regulations.
1. Isolation of Auditory Bulla and Capsule
<ol>
	<li>Euthanize mice in a jar containing a platform above paper towels soaked in isoflurane or sevoflurane until respiratory ventilation ceases for more than a min and then perform cervical dislocation. Be careful to avoid direct contact of mice with the soaked paper towels.</li>
	<li>Make a small transverse incision at the dorsal side of the neck and pull skin apart toward the head and tail using both hands to expose underlying neck muscle tissue.</li>
	<li>Decapitate mice at the cervical region using 14&#160;cm sharp surgical scissors.</li>
	<li>Peel skin completely towards the nose. Cut off all skin together with the snout and incisors.</li>
	<li>Insert scissors into the mouth and cut masseter muscles on both sides.</li>
	<li>Open the jaw carefully and remove the tongue and lower jaw together.</li>
	<li>Using sharp scissors, split skull and skull base into two halves along the midsagittal plane (Figure 1A, B).</li>
	<li>Using forceps, remove the cerebral and cerebellar hemispheres and the brainstem. The auditory bulla and capsule are located lateral to the cerebellum and brainstem. Note that the auditory bulla is further lateral to the auditory capsule (Figure 1C, D).</li>
	<li>Dissect out the bulla and capsule with the surrounding skull bone (Figure 1E).</li>
	<li>Transfer the specimen to a dish containing Phosphate-buffered Saline (PBS) pH 7.4 at RT.</li>
	<li>Under a binocular dissecting microscope, use forceps to pull apart the surrounding bones and scissors to cut the loosened boundary around the bulla and capsule (Figure 1F). The surrounding bones removed are the basioccipital (ventral border), exoccipital (ventro-posterior border), supraoccipital (posterior border), interparietal, parietal (dorsal border), squamosal (dorso-anterior border), alisphenoid (anterior border), and basisphenoid (antero-ventral border) bones. Note that the styliform process (Sp), which supports the tympanic opening of the Eustachian tube 41, is distinct from the styloid process of the temporal bone.</li>
</ol>
2. Isolation of Auditory Ossicles: Malleus, Incus and Stapes&#160;
<ol>
	<li>Malleus
	<ol>
		<li>Using both small scissors and forceps, remove the part of the external auditory canal lateral to the sulcus tympanicus so that the tympanic membrane is visible (Figure 2A, B).</li>
		<li>Remove part of the tympanic membrane and tympanic bone near the malleal processus brevis (orbicular apophysis, see Discussion), both at the ventral (dotted) and posterior (#) walls (Figure 2C). The malleus and tensor tympani muscle should now be exposed (Figure 2D, E).</li>
		<li>Lift the malleus (Figure 2F) and cut the tensor tympani muscle with the beveled edge of a 27 G&#160;needle (Figure 2G). Note that the malleal manubrium firmly attaches to the tympanic membrane, as is seen in other mammals.</li>
		<li>Detach the tympanic membrane carefully from the manubrium, which is fragile. Remove the tympanic bone to reveal the three auditory ossicles.</li>
		<li>Dislocate the malleus from the incus at the ossicular joint (Figure 2H).</li>
		<li>Isolate the malleus by fracturing the anterior process at the goniale.</li>
	</ol>
	</li>
	<li>Incus and Stapes
	<ol>
		<li>Isolate the incus by cutting off the posterior ligament of the incus at the short crus (Figure 3A).</li>
		<li>Isolate the stapes by cutting off the stapedial artery near the stapes with the beveled edge of a 27 G&#160;needle (Figure 3B, C). If necessary, cut the tendon of the stapedial muscle at the muscular process of the stapes with the needle.</li>
		<li>Insert a sewing needle (or a marking pin) into the obturator foramen of the stapes and lift up the stapes. After removing the stapes, the oval window opening should be clearly visible (Figure 3D).</li>
	</ol>
	</li>
</ol>
3. Embedding of Auditory Bulla and Capsule
<ol>
	<li>Preparation for embedding in paraffin blocks
	<ol>
		<li>Isolate the bulla and capsule as described in Section 1.</li>
		<li>Cut the anterior end of the bulla (the styliform process) off with scissors, immerse the bulla and capsule in 4% paraformaldehyde (PFA) in PBS at 4 &#176;C, and allow fixative to enter into the bulla. If air becomes trapped in the bulla, remove it with a needle and syringe. Leave the bulla and capsule in the fixative at 4 &#176;C O/N on a tube rotator. 
		Caution: PFA is toxic and should be handled carefully.</li>
		<li>Wash once with PBS.</li>
		<li>Decalcify bulla and capsule for a week at 4 &#176;C in 10% ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA-2Na), 100 mM Tris base, pH 7.0, in a 2 mL tube. Change the buffer every other day.</li>
		<li>Wash once with PBS. Specimens can be stored in 70% ethanol in water at 4 &#176;C. Optionally, transfer to 70% ethanol through a graded alcohol series (30%, 50%, 70% in water).</li>
		<li>On a tissue processor, dehydrate specimens in a graded series of ethanol solutions (70%, 2x 95%, 3x 100%, each 1 h), clear in xylene (4x, each 1 h&#160;at 40 &#176;C), and infiltrate specimens with molten paraffin wax42. Optionally, substitute xylene with commercial tissue clearing solution (e.g., Histo-Clear).</li>
		<li>Unload specimens from the processor, and remove them from their cassettes.</li>
		<li>On a tissue embedding console system, place specimens into molds filled with molten paraffin wax. Proceed to embedding (Section 4).</li>
	</ol>
	</li>
	<li>Preparation for embedding in frozen blocks (Kawamoto&#39;s film method)43
	<ol>
		<li>Isolate the bulla and capsule as described in Section 1.</li>
		<li>Cut the anterior end of the bulla (the styliform process) off with scissors, immerse the bulla and capsule in fixative (2% or 1% PFA rather than 4% in PBS to preserve antigenicity) at 4 &#176;C. If air is trapped in bulla, remove it using a needle and syringe. Leave the bulla and capsule in the fixative at 4 &#176;C O/N on a tube rotator.</li>
		<li>Wash bulla and capsule quickly in PBS and immediately immerse in liquid cryo-embedding compound at 4 &#176;C.</li>
		<li>Important: Remove air bubbles if any in the middle and outer ear through aspiration by a needle, and by adding the embedding compound with forceps. Proceed to embedding (Section 4).</li>
	</ol>
	</li>
</ol>
4. Sample Orientation and Embedding
NOTE: The whole bulla and capsule must be arranged in a particular orientation during embedding to cut desired sections. The procedures outlined below are used to section the malleus in various orientations.
<ol>
	<li>Longitudinal (parasagittal) sectioning of the malleus
	<ol>
		<li>Put the lateral side of the bulla or external auditory meatus down in warm paraffin (or cryo-embedding compound). Adjust the orientation so that the neck and transversal lamina of the malleus are parallel to the horizontal bottom of the embedding dish (Figure 4A - C). Note that the tympanic membrane is inclined at an angle of approximately 30&#176; to the vertical in the mouse head (Figure 4A; Figure 59 in Kampen44).</li>
	</ol>
	</li>
	<li>Horizontal sectioning of the malleus
	<ol>
		<li>Place the dorsal crest horizontally in warm paraffin (or cryo-embedding compound). Adjust the orientation of the bulla and capsule so that the neck and transversal lamina of the malleus are perpendicular to the bottom of the embedding dish (Figure 4D - F).</li>
	</ol>
	</li>
	<li>Frontal sectioning (cross sections) of manubrium and tympanic membrane 5
	<ol>
		<li>Place the malleal manubrium in warm paraffin (or cryo-embedding compound) such that it is perpendicular to the bottom of the embedding dish.</li>
	</ol>
	</li>
	<li>Cool down the block to temperatures appropriate to harden paraffin wax on a tissue embedding console system (alternatively, use cryo-embedding compound in a dry ice/hexane bath).</li>
	<li>Process tissue block and cut sections using routine procedures. For example, stain paraffin sections with hematoxylin and eosin (H&#38;E), safranin O (for cartilage), or for Tartrate-resistant Acid Phosphatase (TRAP) activity (for osteoclasts) 3, or by immunohistochemistry. Undecalcified cryosections are suitable for bone labeling using fluorochromes 14, alizarin red staining for calcium, and immunofluorescence 42.</li>
</ol>

The authors have nothing to disclose.

Here, we present a method useful to isolate the auditory bulla and capsule in postnatal mice. Prior to P12, tissues are fragile and can become damaged during isolation. After P12, the auditory bulla and capsule can be easily isolated from surrounding tissues. Dissecting the bulla from the head before sectioning has several advantages. First, postnatal cavitation and growth of the auditory bulla occur most actively from P6 onwards and are complete by P14 50. The mesenchymal tissue between the tympanic membrane and cochlear wall is replaced by air through the cavitation process. The resultant air in the middle ear cavity can impede contact between tissues and liquids during fixation, decalcification and embedding. It is easier to remove air from the isolated auditory bulla by cutting off the anterior end (styliform process) rather than trying to do so in the unisolated bulla. Second, orientation of the malleus (and the tympanic membrane) is not vertical in the head. It is therefore easier to section the malleus in desired planes by embedding the isolated auditory bulla and capsule in a given orientation.
Once isolated, auditory bulla and capsules are useful for numerous analyses. For example, high resolution X-ray micro-CT can reveal bone microstructure morphology such as osteogenic capillaries in the malleus 14. The stereofluorescence dissecting microscope is a powerful tool to visualize structures in evaluating reporter mice expressing fluorescent proteins in the middle or inner ear 33. In addition, various in vivo or ex vivo fluorescence labeling methods and whole mount immunofluorescence detection could be undertaken. Light sheet fluorescence microscopy is also useful for three-dimensional analysis 51. Although not described here, diverse anatomical structures associated with the auditory bulla and capsule such as peripheral nerves, blood vessels, and the tympanic membrane in the middle ear can also be evaluated using this protocol.
Note that paraffin sectioning requires decalcification of bone tissues before embedding and therefore does not allow analysis of mineralization. By contrast, the Kawamoto film method 43 used to prepare frozen sections can be performed without decalcification and is suitable for mineralization studies using in vivo bone-labeling techniques or special staining such as Alizarin staining. Cryo-sectioning conditions should be optimized according based on mouse age. For example, a less cool temperature inside the cryostat chamber is recommended for older mouse specimens to minimize damage to sections.
In mouse, the correct term for the prominent semi-spherical protrusion of the malleus is &#34;orbicular apophysis&#34;. Nevertheless, the term &#34;processus brevis&#34; has been widely used to indicate the orbicular apophysis for more than two decades, particularly among mouse developmental biologists 16,20,22-25. &#34;Processus brevis&#34; originally referred to the lateral process (processus lateralis), which differs from the orbicular apophysis. In humans, a lateral process resembling a slight conical projection forms the general line of attachment to the tympanic membrane, extending from the manubrium (not seen in Figure&#160;6B, medial view). In mice, the lateral process is also a projection of the manubrium at the opposite end to the umbo 48. The pars flaccida of the tympanic membrane is above the lateral process of the malleus. Orbicular apophysis is not apparent in the human malleus.

The three auditory ossicles of the middle ear, namely the malleus, incus, and stapes, form a mammalian-specific auditory chain that transmits sound from the tympanic membrane to the inner ear, or cochlea 1,2. Hearing function can be evaluated in mice by measuring Auditory Brainstem Response (ABR) thresholds 3-6, and vibration of the malleus behind the tympanic membrane can be monitored using Laser Doppler Vibrometry (LDV) 7. By combining ABR, LDV, and Distortion Product Otoacoustic Emission (DPOAE) measurements, conductive hearing loss can be discriminated from sensorineural impairment 8.
Animal models of ear conditions are needed, given the importance of hearing and ear health to the well-being of patients of all ages. For example, otitis media is an extremely common ear infection seen in human infants and children, and severe, acute otitis media and its complications can occur if the condition is not treated with appropriate antimicrobials 9. Mouse models of otitis media could prove useful in understanding the pathogenesis and in developing treatments 10,11.
Murine ossicles, which (except for the goniale part of the malleus) are formed by endochondral ossification 12,13, are highly relevant to the study of bone metabolism and morphogenesis. First, their small size allows high-resolution analysis of bones with an intact periosteum using X-ray or fluorescence microscopy 14. Second, aberrant bone metabolism, such as excessive or deficient bone resorption, or impaired interactions among bone cells 15, can be analyzed as a potential contributor to hearing loss 3,4,7. Third, abnormal ossicle morphogenesis is reported in several gene-deficient mice, such as animals lacking Hoxa2 16-19, Msx1 20-22, Prrx1 23, Goosecoid (Gsc) 24,25, Bapx1 13, Tshz1 26, Dusp6 (Mkp3) 27, Noggin (Nog) 28, Fgfr1 29, thyroid hormone receptors (Thra, Thrb) 5, Bcl2 30 and others 1,31, or in mice overexpressing Hoxa2 32. Finally, despite their small size, structures associated with ossicles such as muscles 33 and joints 34,35 are accessible.
Mouse ossicles are smaller than human ossicles, but it is noteworthy that the mouse middle ear is not a miniature version of its human counterpart. For example, in mice, the stapedial artery, which passes through the ring of the stapes, persists throughout life 36, whereas in humans, the embryonic stapedial artery disappears during gestation. In addition, the morphology of the mouse malleus differs from that of the human bone (see Figure 6). In mice, the auditory (tympanic) bulla encloses the air-filled middle ear cavity, whereas in humans, mastoid air cells composed of trabecular bone in the temporal bone houses the ossicles rather than a bulla 37. In both species, the auditory capsule (otic capsule, bony labyrinth) encloses the cochlea and semicircular canals of the inner ear. Comparative and evolutionary biology of the middle ear has been extensively reviewed 38-40.
The protocol provided below first describes how to dissect out the auditory bulla and capsule, which consist primarily of the middle ear and inner ear, respectively. This protocol also demonstrates how to isolate the malleus, incus and stapes from the auditory bulla. Finally, it shows how to orient the auditory bulla and capsule for embedding in preparation for tissue sectioning of auditory ossicles.

<table border="0" cellpadding="0" cellspacing="0" width="823">
	<tbody>
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			<td height="20" style="height:20px;width:240px;">Tools/Equipment</td>
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			<td>can be purchased from other vendors</td>
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			<td height="20" style="height:20px;">14cm surgical scissors</td>
			<td>Fine Science Tools (F.S.T.)</td>
			<td>91400-14</td>
			<td>can be purchased from other vendors</td>
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			<td height="20" style="height:20px;">Extra fine scissors-straight</td>
			<td>Fine Science Tools (F.S.T.)</td>
			<td>14084-08</td>
			<td>can be purchased from other vendors</td>
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			<td height="20" style="height:20px;">Fine Forceps Angled 45&#176;</td>
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			<td>11063-07</td>
			<td>can be purchased from other vendors</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Dissecting microscope</td>
			<td>Nikon</td>
			<td>SMZ800N</td>
			<td>for routine dissection</td>
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			<td height="20" style="height:20px;">Dissecting microscope</td>
			<td>Nikon</td>
			<td>SMZ18</td>
			<td>for movies&#160;</td>
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			<td style="width:259px;">http://www.leicabiosystems.com/histology-equipment/cryostats/details/product/leica-cm3050-s/</td>
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			<td height="20" style="height:20px;">Stainless containers</td>
			<td>Leica</td>
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			<td height="20" style="height:20px;">Cryofilm type IIC</td>
			<td>Leica</td>
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			<td>for Kawamoto&#39;s firm method</td>
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			<td height="20" style="height:20px;">Silane coated slide (New Silane II)</td>
			<td>Muto Pure Chemicals</td>
			<td>511617</td>
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			<td>can be purchased from other vendors</td>
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			<td height="20" style="height:20px;">Tissue processor</td>
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			<td>can be purchased from other vendors</td>
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			<td>can be purchased from other vendors</td>
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			<td height="20" style="height:20px;">Sliding microtome for paraffin</td>
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			<td>REM-710</td>
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			<td>for paraffin section</td>
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			<td height="20" style="height:20px;">Micro-CT</td>
			<td>RIGAKU</td>
			<td>R_mCT2</td>
			<td>http://www.rigaku.com/en</td>
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			<td height="20" style="height:20px;">Fluorescence microscope</td>
			<td>KEYENCE</td>
			<td>BZ-9000</td>
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			<td height="20" style="height:20px;">NaCl</td>
			<td>wako</td>
			<td>191-01665</td>
			<td>for PBS</td>
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			<td height="20" style="height:20px;">KCl</td>
			<td>wako</td>
			<td>285-14</td>
			<td>for PBS</td>
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			<td height="24" style="height:24px;">Na2HPO4 12H2O</td>
			<td>wako</td>
			<td>196-02835</td>
			<td>for PBS</td>
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			<td height="24" style="height:24px;">KH2PO4</td>
			<td>wako</td>
			<td>287-21</td>
			<td>for PBS</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde(EM Grade)</td>
			<td>TAAB</td>
			<td>P001</td>
			<td></td>
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			<td height="20" style="height:20px;">EDTA-2Na</td>
			<td>wako</td>
			<td>15111-45</td>
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			<td height="20" style="height:20px;">Trizma base</td>
			<td>Sigma</td>
			<td>T1503-1KG</td>
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			<td height="20" style="height:20px;">Super Cryoembedding Medium</td>
			<td>Leica</td>
			<td></td>
			<td>for Kawamoto&#39;s firm method</td>
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			<td height="20" style="height:20px;">Dry Ice</td>
			<td>Various</td>
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			<td>for Kawamoto&#39;s firm method</td>
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			<td height="20" style="height:20px;">Hexane</td>
			<td>wako</td>
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			<td height="20" style="height:20px;">Paraffin</td>
			<td>Sakura Finetek</td>
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			<td height="20" style="height:20px;">Histo-Clear</td>
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			<td height="20" style="height:20px;">Hematoxylin&#160;</td>
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</table>

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.

This protocol presents a method to isolate ossicles from the mouse auditory bulla. First, the bulla and capsule are dissected out as a single piece from the skull (Figure 1). The dissected bulla is then used to prepare the malleus (Figure 2) and the incus and stapes (Figure 3). Landmarks of the auditory bulla and capsule are the styliform process at the anterior end of the bulla, the dorsal crest, anterior semicircular canal, and the subarcuate fossa (Figure 1F). Microcomputed tomography (CT) imaging reveals ossicles in the auditory bulla as well as the optimal orientations for longitudinal and horizontal sectioning of those ossicles (Figure 4).
For longitudinal paraffin sectioning of the malleus, the bulla and capsule were decalcified in EDTA at 4 &#176;C for one week, embedded in a paraffin block at the orientation shown in Figure 4 A - C, sectioned at 4 &#181;m, and then stained using H&#38;E. The malleus attached to the tympanic membrane in the auditory bulla revealed ongoing endochondral ossification at P14 (Figure 5A). To visualize new bone formation, calcein (30 &#181;g/g bodyweight) was peritoneally injected into a P20 mouse, and bulla and capsule were isolated 24 h later at P21. The sample without decalcification was embedded frozen and then cryosectioned at 6 &#181;m using an adhesive film based on the method of Kawamoto 43. After nuclear staining with DAPI (4&#39;,6-diamidino-2-phenylindole), the section was observed under a fluorescence microscope. Calcein signals (green) revealed new bone formation in the malleus (m), bulla and capsule (Figure 5B). For horizontal sectioning of the malleus, the auditory bulla isolated from a 5-week-old mouse was embedded frozen without decalcification (for the orientation see Figure 4D - F), cryosectioned at 6 &#181;m using the Kawamoto method, and stained using H&#38;E. Horizontal sectioning of the malleal processus brevis (mPB) also shows the cochlea (Figure 5C).
A medial view of the right auditory ossicles isolated from a P31 mouse shows typical features of the mouse malleus, namely, the &#34;gliding-seagull-wing-like&#34; (or Persian sword-like 45) manubrium, a prominent processus brevis (orbicular apophysis, see Discussion), and the transversal lamina (Figure 6). Note that the anterior process (processus anterior) was fractured in the dissection procedure around the goniale and was separated from the tympanic ring (ectotympanic). This representative sample exhibits an intact incudomalleolar joint between the malleus and incus, whereas the incudostapedial joint is dislocated. Tendinous insertions into the malleal and stapedial muscular processes are detectable (Figure 6A, asterisks).
Figure 6B compares mouse and human auditory ossicles at the same magnification. Species differences, other than size, include the following. The malleal manubrium is wing-like in mice but club-like in humans. The angle between the anatomical axis (or the axis of rotation, the line through the anterior process of the malleus and the short process of the incus) and the manubrium is much smaller in mice and the two are almost parallel, as opposed to nearly perpendicular in humans 6,46-48. In human ossicles, vibrometric studies reveal that the incudo-malleolar joint is mobile rather than functionally fixed 49. The mouse malleus exhibits a wide, thin, and flat transversal lamina not apparent in humans 47. In mice, the processus anterior fuses to membranous bones, namely the goniale and the tympanic ring, while in humans the processus anterior is reduced to a small spicule of bone 41. The stapes of mice and humans also differs: in mice, the anterior crus is curved and the posterior crus is more straight whereas in humans, the anterior crus is more straight than the posterior crus. It is worth noting that the malleus head relative to body size is massively enlarged in species such as the golden mole, demonstrating significant variability in allometric relationships of &#34;the smallest&#34; bones 48.
<img alt="Figure 1" src="/files/ftp_upload/55054/55054fig1.jpg" /> 
Figure 1. Dissection of the Auditory Bulla and Capsule. (A) The skull of a P31 mouse is split into right and left halves. A, anterior; P, posterior; L, left; R, right. (B) Medial surface of the right half of the bisected, skinned head. Cx, cerebral cortex; Cb, cerebellum; Bs, brainstem. D, dorsal; V, ventral. (C) Removal of brain with forceps. (D) Medial view of the auditory capsule in the right skull. The dorsal crest (arrowheads) lies between the middle cranial fossa (mcf) and posterior cranial fossa (pcf) and separates dorso-anterior and ventro-posterior surfaces of the auditory capsule. Scale bar, 2 mm. (E) Higher magnification of auditory bulla and capsule (medial view). Co, cochlea; VII, facial nerve; VIII, vestibulocochlear nerve; AC, anterior (superior) semicircular canal; Sf, subarcuate fossa, which houses the cerebellar paraflocculus. Scale bar, 1 mm. (F) Micrograph of isolated auditory bulla and capsule (medial view). Sp, styliform process. Scale bar, 1 mm. (A - E), P31 mouse. (F), P33 mouse. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55054/55054fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55054/55054fig2.jpg" /> 
Figure 2. Dissection of the Malleus. (A) Ventrolateral view of a right auditory bulla and capsule. The sulcus tympanicus (ST, dashed arrow) is the attachment site of the tympanic membrane. The bone lateral to the ST is part of the external ear, and the bone medial to the ST forms the floor of the middle ear cavity. A, anterior; P, posterior; D, dorsal; V, ventral. (B) View after removal of the external auditory canal to reveal the tympanic membrane (TM) including the pars flaccida (Pf) and pars tensa (Pt). (C) Removal of parts of the tympanic bone (dotted lines and #) near the malleal processus brevis (mPB). m, malleus; mM, malleal manubrium. Arrow, air bubble in the middle ear cavity seen through the tympanic membrane. (D) Exposed malleus. Malleus head is indicated. Dotted line indicates the articular surface of the incus. (E) Tendon of the tensor tympani muscle (TT) attached to the malleus. (F) The tensor tympani is pulled when the malleus is lifted. *, muscular process. (G) Tensor tympani is cut using a needle. (H) Three auditory ossicles after removal of the tympanic membrane. The incudo-malleolar joint is dislocated. m, malleus; i, incus; s, stapes; Go, goniale (fused to the malleus and tympanic ring, TR). All scale bars, 0.5 mm. (A,&#160;H), P33 mouse. (B - G), P31 mouse. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55054/55054fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55054/55054fig3.jpg" /> 
Figure 3. Dissection of the Incus and Stapes. (A) Incus and stapes after removal of the malleus. The stapedial artery (SA) passes through the stapes (s). Dotted line indicates the articular surface of the incus. Note that the short crus (iCB, crus breve) of the incus (i) is fixed by the posterior ligament (not shown). Asterisk, muscular process of the stapes. (B) Stapes after removal of the incus. Needle tip is used to cut the stapedial artery (SA). Arrow, direction of blood flow. Dotted line indicates articular surface of the stapes. (C) The stapedial artery is removed from the stapes. X indicates the cut end of the stapedial artery (SA). (D) The oval window (Ow, fenestra ovalis or fenestra vestibuli) is visible after removal of the stapes. Rw, round window (fenestra rotunda or fenestra cochleae). Scale bars, 0.5 mm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55054/55054fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55054/55054fig4.jpg" /> 
Figure 4. Orienting the Auditory Bulla and Capsule during Embedding for Longitudinal (parasagittal, A - C) and Horizontal (D - E) Sectioning&#160;of the Malleus. (A - C) The neck and transversal lamina of the malleus are placed parallel to the bottom of embedding dish. (A) Side view: micro-CT image to show embedding of the right malleus in the bulla (pseudocolored blue). The malleus and incus are pseudocolored green. Dashed line, the desired cutting plane. Solid line, bottom of embedding dish. m, malleus; arrowheads, dorsal crest. M, medial; L, lateral; D, dorsal; V, ventral. (B) Top view: Micro-CT image. Note that the anterior end of the bulla (styliform process) was removed. i, incus. (C) Top view: micrograph (taken with a color filter). AC, anterior (superior) semicircular canal; Sf, subarcuate fossa; Sp, styliform process. A, anterior; P, posterior; D, dorsal; V, ventral. (D - F) The processus brevis of the malleus is placed perpendicular to the bottom of embedding dish. (D) Side view: Micro-CT image to show embedding of the right malleus. Dashed line, the desired cutting plane. Solid line, bottom of embedding dish. (E) Top view: Micro-CT image. mM, malleal manubrium. (F) Top view: micrograph (taken with a color filter). Scale bars, 1 mm. Micro-CT images were obtained at a voxel resolution of 5 &#956;m, as previously described 7. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55054/55054fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55054/55054fig5.jpg" /> 
Figure 5. Histology. (A) H&#38;E staining. Longitudinal (parasagittal) section of the paraffin-embedded right malleus (m) in the auditory bulla (dotted line) at P14. TM, tympanic membrane. (B) Calcein bone labeling. Longitudinal section of the frozen, undecalcified left malleus (m) in the auditory bulla at P21. Counterstain, DAPI. (C) H&#38;E staining. Horizontal section of the frozen, undecalcified left malleal processus brevis (mPB) in the auditory bulla and capsule (5-week-old mouse). Co, cochlea. Scale bars, 1 mm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55054/55054fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/55054/55054fig6.jpg" /> 
Figure 6. Medial View of Auditory Ossicles. (A) Right auditory ossicles of P31 mouse. A, anterior; P, posterior; D, dorsal; V, ventral. Scale bar, 1 mm. malleus head (Caput mallei, Capitulum mallei); neck (Collum mallei); lamina (transversal lamina); mM (Manubrium mallei); black asterisk (muscular process of the malleus); mPA (Processus anterior, Processus gracilis); mPB (processus brevis); incus body (Corpus incudis); iCB (Crus breve, short crus, short process); iCL (Crus longum, long crus, long process); iPL (Processus lenticularis, lenticular process, Sylvian apophysis); stapes head (Caput stapedis); white asterisk (muscular process of the stapes); sCA (Crus anterius, anterior crus); sCP (Crus posterius, posterior crus); base (Basis stapedis, footplate); sOF (obturator foramen, intercrural foramen). (B) Right auditory ossicles of a 76-year-old human female (Courtesy of Department of Anatomy, Keio University School of Medicine). The ossicles of P31 mouse (lower right) are imaged at the same magnification as that used for human ossicles. Curved arrows indicate the angle between the anatomical axis and the manubrium (dotted lines). Scale bar, 2 mm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55054/55054fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis at JoVE.com

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

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 ...

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29.8K Views.  Keio University School of Medicine. We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis. 

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

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Isolation of Neonatal Extrahepatic Cholangiocytes

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, differences between intra and extrahepatic cholangiocytes, and between neonatal and adult cells, are not well understood.
Methods for the isolation of cholangiocytes from intrahepatic bile ducts are well established1-4. Isolation of extrahepatic ductal cells, especially from the neonate, has not yet been described, although this would be of great benefit in understanding the differences between distinct cholangiocyte populations and in studying diseases such as biliary atresia that appear to target the extrahepatic ducts. Described here is an optimized technique to isolate both neonatal and adult mouse extrahepatic bile duct cells. This technique yields a pure cell population with minimal contamination from mesenchymal cells like fibroblasts.
This method is based on the removal of the extrahepatic ducts and gallbladder, followed by meticulous dissection and scraping to remove fat and fibroblast layers. Structures are embedded in thick layers of collagen and cultured for approximately 3 weeks to allow outgrowth of cholangiocytes in monolayers, which can then be trypsinized and re plated for experimental use.

A technique to isolate cholangiocytes from the extrahepatic bile ducts of neonatal mice is described. The ducts are meticulously dissected, and then cells are isolated by outgrowth in thick collagen gels. This method provides a useful tool for studying extrahepatic bile duct development and pathology.

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

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

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

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

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

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and diseases, such as obesity and fatty liver. Usually, lipids are stored in lipid droplets, which are the multifunctional lipid storage organelles in cells. Lipid droplets vary in size and number in different tissues and under different conditions. It has been reported that lipid droplets are tightly controlled through regulation of its biogenesis and degradation. In Drosophila melanogaster, the oenocyte is an important tissue for lipid metabolism and has been recently identified as a human liver analogue regarding lipid mobilization in response to stress. However, the mechanisms underlying the regulation of lipid droplet metabolism in oenocytes remain elusive. To solve this problem, it is of utmost importance to develop a reliable and sensitive method to directly visualize lipid droplet dynamic changes in oenocytes during development and under stressful conditions. Taking advantage of the lipophilic BODIPY 493/503, a lipid droplet-specific fluorescent dye, described here is a detailed protocol for the dissection and subsequent lipid droplet staining in the oenocytes of Drosophila larvae in response to starvation. This allows for qualitative analysis of lipid droplet dynamics under various conditions by confocal microscopy. Furthermore, this rapid and highly reproducible method can also be used in genetic screens for indentifying novel genetic factors involving lipid droplet metabolism in oenocytes and other tissues.

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Presented here are detailed methods for the dissection and lipid droplet staining of oenocytes in Drosophila larvae using BODIPY 493/503, a lipid droplet-specific fluorescent dye.

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and ...

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Dissection of the Endolymphatic Sac from Mice

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the human phenotypes. Although important progress has been done in the understanding of the development and function of the neurosensory epithelia of the cochlea and vestibula, limited knowledge is available regarding the development, cellular composition, molecular pathways and functional characteristics of the endolymphatic sac. This is, in large part, due to the difficulty of visualizing and microdissecting this tissue, which is an epithelium comprised of only one cell layer. The study presented here describes an approach to access and microdissect the endolymphatic sac from the wild-type mouse inner ear at different ages. The result of a similar dissection is shown in a pendrin-deficient mouse model of enlargement of the vestibular aqueduct. A transgenic mouse with a fluorescent endolymphatic sac is presented. This reporter mouse can be used to readily visualize the endolymphatic sac with limited dissection and determine its size. It can also be used as an educational tool to teach how to dissect the endolymphatic sac. These dissection procedures should facilitate further characterization of this understudied part of the inner ear.

This study describes how to dissect the endolymphatic sac from the inner ear of the mouse at different ages. The result of a similar dissection is shown in an Slc26a4-null mouse model of enlargement of the endolymphatic sac. A transgenic mouse with a fluorescent reporter expressed in the endolymphatic sac is presented as a model to readily visualize the endolymphatic sac, and educational tool.

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the ...