Name: flat mount imaging of mouse skin and its application to the analysis of hair follicle patterning and sensory axon morphology
Uploaded: 2014-06-25T15:00:00.000+00:00
Duration: 13 min 58 s
Description: 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

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>

<ol>
	<li>Burns, T., Breathnach, S., Cox, N., Griffiths, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Rook's Textbook of Dermatology. 8th ed. , (2010).</li><li>Lee, J., Tumbar, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hairy+tale+of+signaling+in+hair+follicle+development+and+cycling.">Hairy tale of signaling in hair follicle development and cycling.</a> Semin. Cell Dev. Biol. 23, 906-916 (2012).</li><li>Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuronal+organization+of+the+retina.">The neuronal organization of the retina.</a> Neuron. 76, 266-280 (2012).</li><li>Chang, H., Nathans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+hair+follicle-associated+structures+to+loss+of+planar+cell+polarity+signaling.">Responses of hair follicle-associated structures to loss of planar cell polarity signaling.</a> Proc. Natl. Acad. Sci. USA. 110, (2013).</li><li>Wallingford, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planar+cell+polarity+and+the+developmental+control+of+cell+behavior+in+vertebrate+embryos.">Planar cell polarity and the developmental control of cell behavior in vertebrate embryos.</a> Annu. Rev. Cell Dev. Biol. 28, 627-653 (2012).</li><li>Guo, N., Hawkins, C., Nathans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frizzled6+controls+hair+patterning+in+mice.">Frizzled6 controls hair patterning in mice.</a> Proc. Natl. Acad. Sci. USA. 101, 9277-9281 (2004).</li><li>Wang, Y., Badea, T., Nathans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Order+from+disorder:+Self-organization+in+mammalian+hair+patterning.">Order from disorder: Self-organization in mammalian hair patterning.</a> Proc. Natl. Acad. Sci. USA. 103, 19800-19805 (2006).</li><li>Wang, Y., Chang, H., Nathans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=When+whorls+collide:+the+development+of+hair+patterns+in+frizzled+6+mutant+mice.">When whorls collide: the development of hair patterns in frizzled 6 mutant mice.</a> Development. 137, 4091-4099 (2010).</li><li>Lumpkin, E. A., Caterina, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+sensory+transduction+in+the+skin.">Mechanisms of sensory transduction in the skin.</a> Nature. 445, 858-865 (2007).</li><li>Wu, H., Williams, J., Nathans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+diversity+of+cutaneous+sensory+afferents+revealed+by+genetically+directed+sparse+labeling.">Morphologic diversity of cutaneous sensory afferents revealed by genetically directed sparse labeling.</a> Elife. 1, (2012).</li><li>Bianchi, N., Depianto, D., McGowan, K., Gu, C., Coulombe, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+the+keratin+17+gene+promoter+to+visualize+live+cells+in+epithelial+appendages+of+mice.">Exploiting the keratin 17 gene promoter to visualize live cells in epithelial appendages of mice.</a> Mol. Cell. Biol. 25, 7249-7259 (2005).</li><li>Alonso, L., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hair+cycle.">The hair cycle.</a> J. Cell Sci. 119, 391-393 (2006).</li><li>Braun, K. M., Niemann, C., Jensen, U. B., Sundberg, J. P., Silva-Vargas, V., Watt, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+stem+cell+proliferation+and+lineage+commitment:+visualisation+of+label-retaining+cells+in+wholemounts+of+mouse+epidermis.">Manipulation of stem cell proliferation and lineage commitment: visualisation of label-retaining cells in wholemounts of mouse epidermis.</a> Development. 30, 5241-5255 (2003).</li><li>Badea, T. C., Wang, Y., Nathans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+noninvasive+genetic/pharmacologic+strategy+for+visualizing+cell+morphology+and+clonal+relationships+in+the+mouse.">A noninvasive genetic/pharmacologic strategy for visualizing cell morphology and clonal relationships in the mouse.</a> J. Neurosci. 23, 2314-2322 (2003).</li><li>Rotolo, T., Smallwood, P. M., Williams, J., Nathans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically-directed,+cell+type-specific+sparse+labeling+for+the+analysis+of+neuronal+morphology.">Genetically-directed, cell type-specific sparse labeling for the analysis of neuronal morphology.</a> PLoS One. 3, (2008).</li><li>Devenport, D., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planar+polarization+in+embryonic+epidermis+orchestrates+global+asymmetric+morphogenesis+of+hair+follicles.">Planar polarization in embryonic epidermis orchestrates global asymmetric morphogenesis of hair follicles.</a> Nat. Cell Biol. 10, 1257-1268 (2008).</li><li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functional+organization+of+cutaneous+low-threshold+mechanosensory+neurons.">The functional organization of cutaneous low-threshold mechanosensory neurons.</a> Cell. 147, 1615-1627 (2011).</li><li>Meyers, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lighting+up+the+senses:+FM1-43+loading+of+sensory+cells+through+nonselective+ion+channels.">Lighting up the senses: FM1-43 loading of sensory cells through nonselective ion channels.</a> J. Neurosci. 23, 4054-4065 (2003).</li><li>Fujiwara, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basement+membrane+of+hair+follicle+stem+cells+is+a+muscle+cell+niche.">The basement membrane of hair follicle stem cells is a muscle cell niche.</a> Cell. 144, 577-589 (2011).</li><li>Orsini, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technique+of+preparation,+study+and+photography+of+benzyl-benzoate+cleared+material+for+embryological+studies.">Technique of preparation, study and photography of benzyl-benzoate cleared material for embryological studies.</a> J. Reprod. Fertil. 3, 283-287 (1962).</li><li>Ke, M. T., Fujimoto, S., Imai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SeeDB:+a+simple+and+morphology-preserving+optical+clearing+agent+for+neuronal+circuit+reconstruction.">SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction.</a> Nat. Neurosci. 16, 1154-1161 (2013).</li><li>Kuwajima, T., Sitko, A. A., Bhansali, P., Jurgens, C., Guido, W., Mason, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ClearT:+a+detergent-+and+solvent-free+clearing+method+for+neuronal+and+non-neuronal+tissue.">ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue.</a> Development. 140, 1364-1368 (2013).</li><li>Hama, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scale:+a+chemical+approach+for+fluorescence+imaging+and+reconstruction+of+transparent+mouse+brain.">Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain.</a> Nat. Neurosci. 14, 1481-1488 (2011).</li><li>Aal Ert&#252;rk, ., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+imaging+of+solvent-cleared+organs+using+3DISCO.">Three-dimensional imaging of solvent-cleared organs using 3DISCO.</a> Nat. Protoc. 7, 1983-1995 (2012).</li><li>Chung, K., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLARITY+for+mapping+the+nervous+system.">CLARITY for mapping the nervous system.</a> Nat. Methods. 10, 508-513 (2013).</li></ol>

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.

<table><tbody><tr><td>5-bromo-4-chloro-indolyl phosphate (BCIP)</td><td>Roche</td><td>11383221001</td><td></td></tr><tr><td>AM1-43</td><td>Biotium</td><td>70024</td><td></td></tr><tr><td>AM4-65</td><td>Biotium</td><td>70039</td><td></td></tr><tr><td>Benzyl alcohol</td><td>Sigma</td><td>402834</td><td></td></tr><tr><td>Benzyl benzoate</td><td>Sigma&amp;nbsp;</td><td>B-6630</td><td></td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td>LSM700</td><td></td></tr><tr><td>Cy3-alpha smooth muscle actin antibody</td><td>Sigma&amp;nbsp;</td><td>C6198</td><td>1:400</td></tr><tr><td>Cytokeratin-8&amp;nbsp;</td><td>Developmental Studies Hybridoma Bank</td><td>TROMA-I-c</td><td>1:500</td></tr><tr><td>Dissecting microscope</td><td></td><td></td><td></td></tr><tr><td>Dissection tools&amp;nbsp;</td><td>Fine Science Tools</td><td></td><td>scissors and forceps</td></tr><tr><td>Electric razor</td><td></td><td></td><td></td></tr><tr><td>Fluoromount G</td><td>EM Sciences</td><td>17984-25</td><td></td></tr><tr><td>Formalin</td><td>Sigma</td><td>HT501320</td><td></td></tr><tr><td>Glass dishes</td><td>Pyrex&amp;nbsp;</td><td></td><td>6 cm and 10 cm diameter</td></tr><tr><td>Glass plates</td><td>Amersham Biosciences</td><td>SE202P-10</td><td>10 cm x 8 cm x 1 mm</td></tr><tr><td>Hair remover&amp;nbsp;</td><td>Nair</td><td></td><td></td></tr><tr><td>Horizontal rotating platform&amp;nbsp;</td><td>Hoefer</td><td>PR250 Orbital shaker</td><td></td></tr><tr><td>Insect pins</td><td>Fine Science Tools&amp;nbsp;</td><td>26002-20</td><td></td></tr><tr><td>Ketamine/xylazine</td><td>Sigma</td><td>K113</td><td></td></tr><tr><td>Nitroblue tetrazolium (NBT)</td><td>Roche&amp;nbsp;</td><td>11383213001</td><td></td></tr><tr><td>Oil Red O</td><td>Sigma</td><td>O0625</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma&amp;nbsp;</td><td>P6148</td><td></td></tr><tr><td>Razor Blades</td><td>VWR</td><td>55411-055</td><td></td></tr><tr><td>Secondary antibodies&amp;nbsp;</td><td>Invitrogen</td><td></td><td>Alexa-dye conjugated&amp;nbsp;</td></tr><tr><td>Sylgard-184</td><td>Fisher Scientific</td><td>NC9020938</td><td></td></tr><tr><td>Tissue culture plastic dishes</td><td></td><td></td><td>10 cm diameter</td></tr><tr><td>Tissue culture plates</td><td></td><td></td><td>6- and 12-well&amp;nbsp;</td></tr></tbody></table>

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

21.4K Views.  Howard Hughes Medical Institute, Johns Hopkins University School of Medicine.  Zoogdierhuid bevat een divers scala aan structuren - zoals haarfollikels en zenuwuiteinden - die onderscheidend patronen van ruimtelijke organisatie vertonen. Het analyseren van de huid als een platte berg maakt gebruik van de 2-dimensionale geometrie van dit weefsel tot volledige dikte van hoge-resolutie afbeeldingen van de huid structuren te produceren. 

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

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

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 ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Developmental Biology

Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching morphogenesis and patterning, as well as immune cell distribution at a cellular level. The analysis of peripheral nerve and blood vessel anatomical structures in adult tissues provides some insights into the understanding of functional neuro-vascular wiring and neuro-vascular degeneration in pathological conditions such as wound healing. As a highly informative model system, we have focused our studies on adult ear skin, which is readily accessible for dissection. Our simple and reproducible protocol provides an accurate depiction of the cellular components in the entire skin, such as peripheral nerves (sensory axons, sympathetic axons, and Schwann cells), blood vessels (endothelial cells and vascular smooth muscle cells), and inflammatory cells. We believe this protocol will pave the way to investigate morphological abnormalities in peripheral nerves and blood vessels as well as the inflammation in the adult ear skin under different pathological conditions.

Here, we describe a high resolution whole-mount imaging method in the entire adult mouse ear skin, which enables us to visualize branching morphogenesis and patterning of peripheral nerves and blood vessels, as well as immune cell distribution.

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching ...

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

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 ...

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 ...

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

Here, we describe a protocol for visualizing motor neuron projection and axon arborization in transgenic Hb9::GFP mouse embryos. After immunostaining for motor neurons, we used light sheet fluorescence microscopy to image embryos for subsequent quantitative analysis. This protocol is applicable to other neuron navigation processes in the central nervous system.

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in ...

Non-Invasive Visualization of Nailbed Microvascular Morphology in Mice Using Capillaroscopy

Imaging microcapillary networks of the skin in humans using nailfold capillaroscopy (NFC) has underscored the importance of microcirculation as a target organ system in critical systemic illnesses. Nailfold capillaroscopy is applied clinically to detect peripheral microvascular dysfunction and abnormalities in a range of systemic conditions, including rheumatic, cardiac, ocular (e.g., glaucoma), and endocrine disorders (e.g., hypertension and diabetes mellitus). NFC is useful not only in detecting peripheral systemic microvasculature disruption but also in assessing drug efficacy. However, translating clinical NFC findings to animal disease models can be challenging. Detecting microvascular dysfunction or abnormalities in animals is often invasive (e.g., endoscopic), carried out ex vivo (e.g., post-mortem imaging of tissues), or expensive, requiring specialized equipment such as those used in microcomputed tomography and photoacoustic imaging techniques. Developing quick, non-invasive, and inexpensive techniques to image peripheral microvasculature in animal models of disease is warranted to decrease research expenses and increase translatability to the clinic.
Capillaroscopy has previously been used to visualize the nailfold microvasculature in animal models, including in guinea pigs and mice, thus demonstrating the capability of capillaroscopy as a non-invasive imaging tool in animal models. This study provides a protocol that applies capillaroscopy to a mouse nailbed, allowing researchers to easily and inexpensively assess the morphology of its microvasculature. Representative images of typical nailbed microvascular architecture in wild-type mice using two commonly used laboratory strains, SV129/S6 and C57/B6J, are provided. Further studies using this method are essential for applying nailbed capillaroscopy to a wide range of mouse disease models with peripheral microvascular abnormalities.

Capillaroscopy is an accessible tool for direct, inexpensive, and non-invasive visualization of microvasculature. The goal of this protocol is to enable researchers to use capillaroscopy for the visualization of peripheral microvascular morphology in the nailbeds of mice.

Imaging microcapillary networks of the skin in humans using nailfold capillaroscopy (NFC) has underscored the importance of microcirculation as a target ...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video