The authors thank Dr. Amir Rattner for helpful comments on the manuscript. Supported by the Howard Hughes Medical Institute.

This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocol MO11M29 of the Johns Hopkins Medical Institutions. Consult your local Institutional Animal Care and Use Committee guidelines for approved methods of euthanasia. Wear gloves, lab coat, and safety glasses when handling aldehyde fixatives or organic solvents.
1. Preparation of Materials
<ol>
	<li>Pouring Sylgard Plates
	<ol>
		<li>Follow the manufacturer&#39;s instructions, mix the curing agent with the base, and stir with a plastic rod.</li>
		<li>Pipette ~35 ml liquid Sylgard per 10 cm diameter tissue culture dish. Tissue culture dishes are deeper than the standard bacterial dishes; this provides vertical clearance for the insect pins.</li>
		<li>Place the dishes on a horizontal surface at room temperature overnight. Bubbles formed during the mixing will disappear.</li>
		<li>Following polymerization, store the Sylgard plates at room temperature. 
		NOTE: Sylgard plates can be stored at room temperature for years and re-used multiple times until the Sylgard detaches from the plate.</li>
	</ol>
	</li>
	<li>Solutions
	<ol>
		<li>Benzyl benzoate and benzyl alcohol (BBBA). Mix 2 volumes benzyl benzoate and 1 volume benzyl alcohol. Store in the dark in a glass bottle with a glass stopper or Teflon top. 
		CAUTION: Used BBBA should be appropriately handled as organic solvent waste. Do not allow BBBA to contact plastic.</li>
		<li>Phosphate buffered saline (PBS). Add 4 g PFA to 80 ml water, add 0.1 ml 1 N NaOH, stir on a hot plate at ~70 &#176;C for ~5 min until the PFA is completely dissolved, and then add 10 ml 10x PBS and bring the volume to 100 ml with water. 
		CAUTION: Inhalation of formaldehyde/PFA and BBBA vapors should be minimized by keeping these solutions in covered containers. Formaldehyde/PFA is a carcinogen.</li>
		<li>Oil Red O. Prepare a 0.5% stock in isopropanol. Immediately before use dilute with water to a final concentration of 0.3% Oil Red O, and then filter through a 0.2 &#181;m filter.</li>
	</ol>
	</li>
</ol>
2. Skin Dissection, Fixation, and Clearing
<ol>
	<li>Dorsal Skin for Immunostaining and Melanin Imaging
	<ol>
		<li>For very young mice (e.g., fetuses and postnatal day (P)0-P3 pups), euthanize the mice by isoflurane inhalation. Euthanize older mice by isoflurane inhalation or by ketamine/xylazine injection followed by cervical dislocation.</li>
		<li>Remove the hair with an electric razor and hair removal gel. For mice younger than P8, this step can be skipped.</li>
		<li>Use a razor blade to make a horizontal cut from the base of the tail along each flank, passing on the dorsal side of the base of each limb, proceeding lateral to the ears and ending at the nose.</li>
		<li>Gently peel the skin from the underlying tissue proceeding from posterior to anterior by holding the skin between gloved fingers so that it is not pinched by forceps. When peeling the skin at the level of the ears, first cut off the ears with a pair of scissors and proceed especially slowly as the skin can tear at this location.</li>
		<li>From this point onward, orient the skin with the inside facing up. Pin the skin to Sylgard with ~20 insect pins evenly spaced around the periphery; do not over-stretch the skin (Figure 2B). Cover the skin with 10-20 ml of PBS.</li>
		<li>Dissect away the skin-associated fat and connective tissue using angled or curved forceps with the pointed end of the forceps facing horizontally to minimize the risk that the forceps will penetrate the skin. Extrude any air bubbles that have been trapped beneath the skin by gently rolling a cotton-tipped applicator over the skin surface. 
		NOTE: If the skin is going to be used for flat mount immunostaining or histochemistry, remove as much of the connective tissue as possible; if the skin is going to be used to visualize hair follicles based on melanin pigmentation, less complete removal of connective tissue is satisfactory. Prenatal skin requires minimal &#8220;cleaning&#8221; of adherent connective tissue.</li>
		<li>Fix the pinned skin by adding 20 ml of freshly prepared 4% PFA/PBS or 10% buffered formalin per 10 cm Sylgard plate (if the skin will be used for melanin imaging or alkaline phosphatase (AP) histochemistry) or 20 ml of freshly prepared 2% PFA/PBS (if the skin will be used for immunostaining or visualizing transgenic Keratin (Krt)17-GFP11), and gently rotate overnight in a cold room. The next day, wash in PBS for &#62;10 min. 
		NOTE: Standard formalin is 37% formaldehyde; therefore, &#8220;10% formalin&#8221; is 3.7% formaldehyde.</li>
		<li>For AP staining and immunostaining, continue in Section 3. For melanin imaging, dehydrate the skin through a graded ethanol series (70%, 95%, 100%), one day for each step, with gentle horizontal rotation at room temperature. Remove residual connective tissues.</li>
		<li>With the skin in 100% ethanol, remove the insect pins and, if desired, trim the skin with scissors to remove the most peripheral 1-2 mm of skin with the pin holes.</li>
		<li>Transfer the skin to a 10 cm diameter glass dish, place two glass microscope slides on the skin to prevent it from curling, and add 20 ml of BBBA. BBBA rapidly hardens tissue so it is important for the skin to be flattened under the weight of the glass slides before adding the BBBA.</li>
		<li>Over the next several hours, lift the slides off the skin for a few seconds to allow the BBBA to wash over the skin. 
		NOTE: Wear gloves when working with BBBA. Depending on the age and location of the skin there can be as much as 30% shrinkage in BBBA.</li>
	</ol>
	</li>
	<li>Foot Skin for the Analysis of Hair Follicle Patterning 
	NOTE: The range of ages typically examined is P1-P8.
	
	<ol>
		<li>After euthanasia, cut off the feet above the ankle joint and make a single straight cut along the ventral surface through the soles of the feet.</li>
		<li>Gently peel the skin from the underlying tissue, proceeding in a proximal to distal direction.</li>
		<li>Cut the digits to release the skin and pin it to Sylgard with the inside surface facing up (Figure 2C). There is minimal connective tissue on the foot skin.</li>
		<li>Transgenic Krt17-GFP skin is now ready for imaging. For melanin imaging proceed with fixation, dehydration and BBBA clearing as described in section 2.1.</li>
	</ol>
	</li>
	<li>Foot Skin for Visualizing Sebaceous Glands
	<ol>
		<li>After euthanasia, remove the hair by rubbing hair remover gel over the skin surface with a gloved finger and thumb; wait for 10 min. 
		NOTE: Do not use an electric razor, which will damage the delicate foot skin.</li>
		<li>Wash the skin surface with tap water, dissect and pin the skin to Sylgard as described in section 2.2.</li>
		<li>Fix with 1% PFA/PBS for 30 min at room temperature and then wash in PBS. 
		NOTE: For visualizing sebaceous glands on the feet, which develop after the first postnatal week, the optimal age for analysis with Oil Red O is P21, because this marks the nadir of hair pigmentation during the first hair cycle12, and therefore the sebaceous glands can be seen more readily. With albino mice this analysis could be performed at any age. To obtain albino progeny cross pigmented mutant mice to a tyrosinase mutant line such as C57BL/6J-Tyrc-2J .</li>
	</ol>
	</li>
	<li>Tail Skin Preparation for the Analysis of Melanin Distribution, Hair Follicle Orientation, and Sebaceous Gland Visualization
	<ol>
		<li>After euthanasia, make a circular cut around the base of the tail, and then a longitudinal slit that runs the length of the tail along its ventral face.</li>
		<li>Peel off the tail skin starting at the tip by firmly grasping the tip of the tail-bone and/or connective tissue with one pair of forceps and the skin with a second pair of forceps. Pin the tail skin to Sylgard.</li>
		<li>For the analysis of melanin distribution and hair follicle orientation, fix and process the skin as described in section 2.1. For analysis of sebaceous glands, cut the tail skin into a series of segments ~0.5 to 1 cm in length prior to fixation and incubate the skin pieces in PBS/5 mM EDTA overnight at 37 &#176;C to weaken the dermis-epidermis adhesion13.</li>
		<li>Gently peel the epidermis from the dermis, pin the epidermis to Sylgard (inner face up), and fix it in 4% PFA/PBS for 1 hr at room temperature. Wash with PBS, and stain with Oil Red O as described below. 
		NOTE: An electric razor and/or hair removal gel can be used prior to dissecting older tail skin (e.g., P21), but this treatment is optional since the hair on the tail is not as dense as elsewhere on the body.</li>
	</ol>
	</li>
</ol>
3. Staining Reactions
<ol>
	<li>Human Placental Alkaline Phosphatase (AP) Histochemistry to Visualize Genetically-labeled Axon Arbors10,14,15 (Figures 3A,B)
	<ol>
		<li>After PFA fixation on Sylgard, place fixed skins in a 10 cm glass dish and submerge under PBS/1 mM MgCl2. Gently rotate the dish in a water bath at 70 &#176;C for 90 min to destroy endogenous phosphatase activity.</li>
		<li>Visualize AP reporter activity histochemically with a 4-48 hr incubation at room temperature in 0.1 M Tris, pH 9.5, 0.1 M NaCl, 50 mM MgCl2, 0.34 &#181;g/ml NBT, and 0.175 &#181;g/ml BCIP, typically using 10-15 ml of staining solution per P21 dorsal skin. Monitor the reaction under the dissecting microscope, taking care not to let it proceed beyond the optimal time, as judged by visual inspection of axon staining intensity relative to nonspecific NBT deposition.</li>
		<li>Stop the reaction by washing with three changes of PBS/0.1% Tween-20 over the course of 1 hr, re-pin the skins to Sylgard, dehydrate the skins through an ethanol series, and then transfer the skins to a 10 cm dish with BBBA. 
		NOTE: BBBA slowly dissolves the NBT precipitate. During the first few hours in BBBA, a fine background of precipitated NBT will dissolve; as a result, the BBBA solution will darken, and the skin will lighten. Change to fresh BBBA after a few hours.</li>
		<li>After imaging the AP-stained skin, transfer it to ethanol for &#62;1 hr at room temperature and then store it in fresh ethanol at -20 &#176;C. AP-stained skins can be stored in this way for many months with no loss of signal.</li>
	</ol>
	</li>
	<li>Oil Red O Staining to Visualize Sebaceous Glands4 (Figures 3C-F)
	<ol>
		<li>Remove insect pins and transfer the lightly fixed foot or tail skins from the Sylgard plate to the wells of a 6-well tissue culture plate, with up to two skin samples per well. Wash with 60:40 isopropanol:water for 5 min at room temperature.</li>
		<li>Stain in 2 ml 0.3% Oil Red O in 60:40 isopropanol:water (see section 1.2) at room temperature for 2 hr.</li>
		<li>Wash with 60:40 isopropanol:water for 5 min at room temperature, followed by a water wash. Carefully trim away the edges of the skin, including the pin holes, with fine scissors so that the surface of the skin can be made as flat as possible.</li>
		<li>Place the skin on a slide with the outer surface facing up, add a few drops of water or aqueous mounting medium, cover the skin with a coverslip, and then gently tape the coverslip to the slide to further flatten the skin.</li>
	</ol>
	</li>
	<li>Flat Mount Immunostaining4,16,17 (Figures 3G,H)
	<ol>
		<li>Cut a rectangle of PFA fixed skin (e.g. 1 cm x 0.5 cm), marking its orientation with an asymmetric cut (e.g. clip the anterior right corner). Perform the incubation and wash steps with gentle rotation on a rotating horizontal platform. In the wells of a 6- or 12-well tissue culture plate, wash several times with PBS to remove residual PFA, wash with ~10 changes of PBS with 0.3% Triton X-100 (0.3% PBST) over 5&#8211;8 hr at room temperature to permeabilize the skin.</li>
		<li>Incubate with the primary antibody in 0.3% PBST with 5% goat serum and 20% DMSO for five days at room temperature. Cover the wells of the plate with clear tape or adhesive plastic to eliminate evaporative losses.</li>
		<li>Wash with 10-15 changes of 0.3% PBST over 5&#8211;8 hr and incubate with secondary antibody in 0.3% PBST with 5% goat serum and 20% DMSO for 3 days at room temperature.</li>
		<li>Wash with 10-15 changes of 0.3% PBST over 5&#8211;8 hr.</li>
		<li>Dehydrate in 25%, 50%, and 75% methanol for 5 min each and then 3x in 100% methanol for 20 min each.</li>
		<li>Clear in BBBA overnight at room temperature. 
		NOTE: Flat mount immunostaining of skin younger than ~P1 can be performed as described above but with shorter incubation and wash times. For example, P1 skin can be immunostained with an overnight incubation in primary antibody and a several hour incubation in secondary antibody, and with intervening washes of only 2-3 hr in 0.3% PBST. As noted above, younger skins are also sufficiently thin that they can be imaged without BBBA clearing.</li>
	</ol>
	</li>
	<li>Visualizing Merkel Cell Clusters with Fluorescent Nerve Terminal Dye Uptake (Figures 3I-L)
	 AMI-43 (green) and AM4-65 (red) are fixable fluorescent dyes that enter cells via non-selective cation channel18. In the living skin these dyes are selectively taken up and concentrated in Merkel cells.
	<ol>
		<li>Dissolve 1 mg AM1-43 or AM4-65 in 2 ml PBS and store in aliquots at -80 &#176;C.</li>
		<li>Inject newborn pups subcutaneously with 2-10 &#181;g dye per gram body weight using a 29 G needle.</li>
		<li>One day later, euthanize the mice and dissect the dorsal skins.</li>
		<li>Fix the skins in 4% PFA/PBS at 4 &#176;C overnight, wash with PBS, and mount the skin as described in section 3.2.</li>
	</ol>
	</li>
</ol>
4. Imaging and Image Analysis
<ol>
	<li>Imaging Hair Follicle Orientation (Figure 4)
	<ol>
		<li>Use a dissecting microscope to image dorsal, foot, or tail skins that are submerged in BBBA, flattened beneath a glass slide, and still in the glass dish - this approach minimizes BBBA contamination of the microscope.</li>
		<li>Illuminate the dish from below. To minimize spatial variation in light intensity across the field of view, elevate the glass dish to a height ~10 cm above the standard working surface of the dissecting microscope and place a diffusing plastic or glass plate over the light source.</li>
		<li>Return the skin to 100% ethanol for long-term storage at -20 &#176;C. 
		NOTE: Leaving the skin in BBBA for more than a day causes the melanin granules to break apart. Once the skin has been BBBA treated it will harden and remain flat from that point onward. Different skins can be coded with a series of notches at their anterior and/or posterior ends, so that they can be store together, to save space in the freezer.</li>
	</ol>
	</li>
	<li>Imaging and Tracing Axon Arbors (Figures 3A,B)
	<ol>
		<li>Place AP stained skin (section 3.1.3) between two glass plates of the type used for small protein gels. Avoid introducing air bubbles between the plates and the skin, and then carefully wick away any excess BBBA with a paper towel. Place the sandwich of plate-skin-plate on a microscope stage (Figure 2D).</li>
		<li>Use bright-field or DIC illumination with a 10X objective and 2 &#181;m or 5 &#181;m Z-steps. Use a mechanized X-Y stage to capture a montage of X-Y images that are stitched together to create a single three-dimensional grey-scale data set. 
		NOTE: For a single large axon arbor, this data set can be as large as 5 Gb.</li>
		<li>Perform manual, automated, or semi-automated tracing of axon arbors with any of a variety of Software packages. 
		NOTE: Software such as Neuromantic (http://www.reading.ac.uk/neuromantic/) is a free neuronal reconstruction tool that runs in a PC environment and is relatively easy to master10.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Mastery of the dissection methods described above requires only patience, a steady hand, and a few good dissection tools. The dorsal skin dissection is relatively easy, but the tail and foot skin dissections &#8211; especially at early postnatal ages &#8211; are more challenging. At early prenatal ages (e.g., before E15), the skin is difficult to remove without tearing it. Conveniently, for many studies of growth and patterning of skin structures in mice, the events of interest occur postnatally, as seen for exa...

The skin is one of the largest organs in the body, with important functions in somato-sensation, insulation/thermoregulation, and immune defense1. Understanding the molecular and cellular basis of skin development and function has been of longstanding interest because of the fundamental importance of skin as a biological system and its relevance to dermatology. Mammalian skin contains a variety of multicellular structures, including stratified layers of keratinocytes, dermal connective tissue, several types of hair follicles, sebaceous glands, arrector pili muscles, blood vessels, and at least a dozen distinct classes of afferent (sensory) and efferent nerve fibers (Figure 1). Different regions of the body are associated with characteristically different types of skin. In most mammals, nearly the entire body surface is covered with skin that is densely packed with hair follicles. [Humans and naked mole rats constitute exceptions to this pattern.] Hair is missing from the palmar surfaces of the hands and feet, which are also associated with specialized epidermal patterns (dermatoglyphs), exocrine glands, and sensory nerve endings. The cellular and molecular events that control the growth, differentiation, and spatial arrangement of cells within the hair follicle are of special interest as each follicle exhibits, in miniature, many of the central features of organogenesis2. These features include the existence of stem cells and a stem cell niche, precisely choreographed cell migrations, and the assembly of multicellular structures from embryologically distinct components.
This article describes methods for dissecting, fixing, labeling, and imaging mouse skin as an intact two-dimensional sheet, referred to as a &#8220;whole mount&#8221; or &#8220;flat mount&#8221; preparation. Since mouse skin is relatively thin, it is possible to image through the full thickness of flattened skin using conventional confocal microscopy. The flat mount approach to imaging mammalian skin is technically advantageous because it bypasses the need for physical sectioning, thereby allowing structures to be reconstructed entirely by optical sectioning. Since nearly the entire skin is processed as a single object, the flat mount approach also facilitates the imaging of multiple regions of the body surface while preserving information about position and orientation relative to the body axes. Finally, structures within the skin are typically present in patterns that are repeated at regular intervals, thus facilitating the collection of images from multiple representatives of a given structure. These characteristics are familiar to neurobiologists who work on the retina, a two-dimensional part of the central nervous system that enjoys analogous advantages for studies of neuronal morphology3.
The flat mount approach described here is of special utility for studying structures that exhibit spatial organization on a relatively large scale within the two-dimensional plane of the skin. One example of large-scale spatial organization is the coordinated polarity of hair follicles and hair follicle-associated structures - Merkel cell clusters, arrector pili muscles, sebaceous glands, and nerve endings4. Hair follicles are oriented at an angle with respect to the plane of the skin, and the component of the follicle vector that lies within the 2-dimensional plane of the skin generally exhibits an orientation with respect to the body axes that is precisely determined for each position on the body. For example, hair follicles on the back point from rostral to caudal and hair on the dorsal surface of the feet point from proximal to distal. Hair follicle orientation is controlled by planar cell polarity signaling (PCP; also called tissue polarity5). This signaling system was discovered in Drosophila where a small set of core PCP genes was found to control the orientation of cuticular hairs and bristles. Three mammalian orthologues of core PCP genes - frizzled homolog 6 (Fzd6, also referred to as Fz6), cadherin EGF LAG seven-pass G-type receptor 1 (Celsr1), and vang-like 2 (Vangl2) - play analogous roles in mammalian skin, coordinating the orientations of hair follicles with the body axes. Studies of Fz6 knockout mice (Fzd6tm1Nat, hereafter referred to as Fz6-/-) show that the primary defect in the absence of PCP signaling is an initial randomization or disorganization of hair follicle orientation, with no effect on the intrinsic structure of the follicles6-8. A second non-PCP system acts later to promote local alignment of nearby follicles, which leads to the production of large-scale hair patterns such as whorls and tufts.
A second example of large-scale spatial organization within the skin is seen in the morphologies of sensory axon arbors. Sensory neurons that innervate the skin have their cell bodies in the dorsal root and trigeminal ganglia. These neurons detect temperature, pain, itch, and various types of mechanical deformations impinging on the skin and hair9. They can be divided into subtypes based on axon diameter and conduction velocity, terminal nerve ending structure, and the patterns of expression of receptors, channels, and other molecules. Because of the high density of innervation within the skin, analyses that involve visualizing all axons (e.g., anti-neurofilament immunostaining) or even all axons of a single class (as seen when a single cell type is marked by expression of a fluorescent reporter) generally reveals a dense superposition of axons that makes it impossible to define the morphology of an individual arbor. To circumvent this problem, we have used extremely sparse genetically-directed labeling to produce dorsal skin samples in which individual well-isolated axon arbors are visualized by expression of a histochemical reporter, human placental alkaline phosphatase10. This approach allows the unambiguous visualization of individual axon arbor morphologies and a definition of somatosensory neuron types based on morphologic criteria.

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			<td height="21" style="height:21px;">5-bromo-4-chloro-indolyl phosphate (BCIP)</td>
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			<td>11383221001</td>
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			<td>402834</td>
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			<td>Sigma&#160;</td>
			<td>C6198</td>
			<td>1:400</td>
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			<td height="21" style="height:21px;">Cytokeratin-8&#160;</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>TROMA-I-c</td>
			<td>1:500</td>
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			<td></td>
			<td></td>
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			<td height="21" style="height:21px;">Dissection tools&#160;</td>
			<td>Fine Science Tools</td>
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			<td>scissors and forceps</td>
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			<td>EM Sciences</td>
			<td>17984-25</td>
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			<td>Sigma</td>
			<td>HT501320</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Glass dishes</td>
			<td>Pyrex&#160;</td>
			<td></td>
			<td>6 cm and 10 cm diameter</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Glass plates</td>
			<td>Amersham Biosciences</td>
			<td>SE202P-10</td>
			<td>10 cm x 8 cm x 1 mm</td>
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			<td height="21" style="height:21px;">Hair remover&#160;</td>
			<td>Nair</td>
			<td></td>
			<td></td>
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			<td height="21" style="height:21px;">Horizontal rotating platform&#160;</td>
			<td style="width:256px;">Hoefer</td>
			<td style="width:145px;">PR250 Orbital shaker</td>
			<td></td>
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			<td height="21" style="height:21px;">Insect pins</td>
			<td>Fine Science Tools&#160;</td>
			<td>26002-20</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Ketamine/xylazine</td>
			<td>Sigma</td>
			<td>K113</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Nitroblue tetrazolium (NBT)</td>
			<td>Roche&#160;</td>
			<td>11383213001</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Oil Red O</td>
			<td>Sigma</td>
			<td>O0625</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma&#160;</td>
			<td>P6148</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Razor Blades</td>
			<td style="width:256px;">VWR</td>
			<td style="width:145px;">55411-055</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Secondary antibodies&#160;</td>
			<td>Invitrogen</td>
			<td></td>
			<td>Alexa-dye conjugated&#160;</td>
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			<td height="21" style="height:21px;">Sylgard-184</td>
			<td>Fisher Scientific</td>
			<td>NC9020938</td>
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			<td height="21" style="height:21px;">Tissue culture plastic dishes</td>
			<td></td>
			<td></td>
			<td>10 cm diameter</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Tissue culture plates</td>
			<td></td>
			<td></td>
			<td>6- and 12-well&#160;</td>
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flat mount imaging of mouse skin and its application to the analysis of hair follicle patterning and sensory axon morphology

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale - as seen, for example, in the orderly arrangement of cell types within a single hair follicle - and on a macroscopic scale - as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.

Brightfield imaging of skin flatmounts can be used to image cutaneous sensory afferents (Figure 3A10) and hair follicle patterns based on melanin pigmentation (Figure 4). Confocal imaging of skin flatmounts can be used to define the geometry of (1) Merkel cell clusters, visualized with anti-cytokeratin-6 or with AM dye uptake (Figures 3I-L), (2) arrector pili muscles, visualized with anti-smooth muscle actin (Figures 3G,H), (3) sebaceous gland...

Watch this Scientific Journal Video about Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology at JoVE.com

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel ...

flat-mount-imaging-mouse-skin-its-application-to-analysis-hair

Research

JoVE Journal

Neuroscience

21.3K vues.  Howard Hughes Medical Institute, Johns Hopkins University School of Medicine.  La peau des mammifères contient un large éventail de structures - comme les follicules pileux et les terminaisons nerveuses - qui présentent des modèles distinctifs de l'organisation spatiale. Analyser la peau comme un support plat profite de la géométrie à 2 dimensions de ce tissu pour produire toute l'épaisseur des images haute résolution des structures de la peau. 

Vidéo: Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

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

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

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

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

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

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

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

Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicle Lanceolate Endings

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The technique is very cost-effective and easily undertaken with materials commonly found in most electrophysiology laboratories, or easily purchased. The dissection is simple and fast, with the mechanical displacement provided by a generic electroceramic wafer controlled by proprietary software. The same software also records and analyses the electroneurogram output. The recording of the evoked nerve activity is through a commercial differential amplifier connected to fire-polished standard glass microelectrodes. Helpful tips are given for improving the quality of the preparation, the stimulation and the recording conditions to optimize recording quality. The system is suitable for assaying the electrophysiological and optical properties of lanceolate terminals of palisade endings of hair follicles, as well as the outcomes from their pharmacological and/or genetic manipulation. An example of combining electrical recording with mechanical stimulation and labeling with a styryl pyridinium vital dye is given.

A simple and novel technique for recording afferent discharge due to mechanical stimulation of lanceolate terminals of palisade endings innervating mouse ear skin hair follicles is presented.

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The ...

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs).

This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation.

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...