Name: whole-mount confocal microscopy for adult ear skin: a model system to study neuro-vascular branching morphogenesis and immune cell distribution
Uploaded: 2018-03-29T15:00:00.000+00:00
Duration: 8 min 42 s
Description: Watch this Scientific Journal Video about Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution at JoVE.com

We thank K. Gill for the laboratory management and technical support, J. Hawkins and the staff of National Institutes of Health (NIH) Building 50 animal facility for the assistance with mouse care, and R. Reed and F. Baldrey for administrative assistance. Thanks also to S. Motegi and M. Udey for sharing their ear skin dissection protocol, N. Burns for editorial help, and members of the Laboratory of Stem Cell and Neuro-Vascular Biology for technical help and thoughtful discussion. T. Yamazaki was supported by the Japan Society for the Promotion of Science (JSPS) NIH-KAITOKU. This work was supported by the Intramural Research Program of the National Heart, Lung, and Blood Institute (HL005702-11 to Y.M.)

All experiments in this section were performed under approval from the National Heart, Lung, and Blood Institute (NHLBI) Animal Care and Use Committee.
1. Adult Mouse Ear Skin Collection
<ol>
	<li>Euthanize adult mice by carbon dioxide (CO2) exposure in a closed chamber and then confirm the euthanasia by cervical dislocation. 
	NOTE: The experiment follows the National Institutes of Health (NIH) guideline for the euthanasia method.</li>
	<li>Dissect the ear from the base and place it in a 35 x 10 mm2 Petri dish containing 2 mL of Hank&#39;s Balanced Salt Solution (HBSS). Briefly trim hairs with scissors.</li>
	<li>Peel the posterior skin and anterior skin away carefully from the intervening cartilage. 
	NOTE: Cartilage attaches to the anterior skin.</li>
	<li>Transfer the posterior and anterior skin separately to a 24 well plate containing 1 mL of ice-cold fresh 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) per well. Flatten the posterior and anterior skin in the 4% PFA.</li>
	<li>Fix the posterior and anterior skin with cartilage at 4 &#176;C for 1 h.</li>
	<li>Wash the posterior and anterior skin 3 times for 5 min in 1 mL of PBS with gentle mixing on a mixer at room temperature.</li>
	<li>Transfer the posterior and anterior skin with cartilage to the bottom of a 35 x 10 mm2 Petri dish. Cut off the base region, which is folded and has adipose and connective tissues. Peel off the cartilage from anterior skin using fine curved forceps.</li>
	<li>Carefully remove the hairs, adipose tissues, and connective tissues from the inside of the posterior skin using fine curved tweezers. Keep the skin wet with PBS.</li>
</ol>
2. Whole-mount Immunohistochemical Staining of Mouse Ear Skin
NOTE: All experiments in the following sections were performed in accordance with the NIH laboratory safety guidelines.
<ol>
	<li>Prepare the blocking buffer. Filter 10% heat inactivated goat serum (HIGS) diluted in PBS with 0.2% triton X-100 (TX-100) blocking buffer, or 10% donkey serum (DS) diluted in PBS with 0.2% TX-100 blocking buffer, using a 0.45 &#181;m filter unit.</li>
	<li>Transfer the posterior and anterior skin to a 24 well plate containing 1 mL of 10% HIGS blocking buffer or 10% DS blocking buffer per well. Incubate the skin for 30 min with gentle mixing on a mixer at room temperature.</li>
	<li>Prepare the primary antibody solution by diluting the primary antibodies (Table of Materials) in blocking buffer (either 10% HIGS or 10% DS). 
	NOTE: Whole-mout immunohistochemical analysis of adult ear skin with antibodies to pan-neuronal marker neuron-specific class III &#946;-tubulin (Tuj1, rabbit polyclonal IgG or mouse monocloncal IgG2a, 1:500 dilution at the final concentration of 2 &#181;g/mL), pan-endothelial cell marker platelet endothelial cell adhesion molecule 1 (PECAM-1, hamster monoclonal IgG, 1:300 dilution at the final concentration of 3.3 &#181;g/mL), myelin sheath marker myelin basic protein (MBP, rabbit polyclonal IgG, 1:200 dilution at the final concentration of 5 &#181;g/mL) and inflammatory myeloid cell marker CD11b (Rat monoclonal IgG2b, 1:50 dilution at the final concentration of 20 &#181;g/mL) was shown in Figure 1 and Figure 2. The skin was incubated with Cy3-conjugated antibody for vascular smooth muscle cell marker &#945; smooth muscle actin (&#945;SMA) together with secondary antibodies (2.6). The primary antibodies tested by ourselves were listed in Table of Materials. Multiple primary antibodies derived from different species can be mixed simultaneously.</li>
	<li>Transfer the posterior and anterior skin to new well&#160;containing 150 &#181;L of the primary antibodies solution. Incubate the skin with gentle mixing on a mixer at 4 &#176;C overnight.</li>
	<li>On the following day, transfer the posterior and anterior skin to new wells in the 24 well plate or aspirate the blocking buffer containing primary antibodies. Add 1 mL of either 2% HIGS diluted in PBS with 0.2% TX-100 washing buffer or 2% DS diluted in PBS with 0.2% TX-100 washing buffer. Wash the skin with 3 changes of the washing buffer every 15 min with gentle mixing on a mixer at room temperature.</li>
	<li>Prepare the&#160;secondary antibody solution (Table of Materials). Dilute secondary antibodies in the blocking buffer (either 10% HIGS or 10% DS) and filter the blocking buffer containing secondary antibodies through a 0.22 &#181;m polyvinylidene difluoride (PVDF) membrane syringe filter connected to a 1 mL syringe. 
	NOTE: Alexa 488 or 633-conjugated goat anti-rabbit IgG (H+L) or mouse IgG2a for Tuj1, Alexa 647-conjugated goat anti-hamster IgG (H+L) for PECAM-1, Alexa 488-conjugated goat anti-rabbit IgG (H+L) for MBP, and Alexa 594-conjugated rat IgG (H+L) for CD11b were used with 1:250 dilution at the final concentration of 8 &#181;g/mL. The skin was incubated with Cy3-conjugated &#945;SMA antibody (1:500 dilution at the final concentration of 2-3 &#181;g/mL) in combination with these secondary antibodies.</li>
	<li>Centrifuge the solution at 13,000 x g for 10 min to remove aggregated particles of the secondary antibodies from the blocking buffer. 
	NOTE: Different fluorescent conjugated secondary antibodies derived from different species can be mixed simultaneously.</li>
	<li>Transfer the posterior and anterior skin to well&#160;containing 150 &#181;L of the secondary antibodies solution. Wrap the 24 well plate in aluminium foil to avoid light and incubate the skin for 1 h with gentle mixing on a mixer at room temperature.</li>
	<li>Transfer the posterior and anterior skin to new wells in the 24 well plate or aspirate the secondary antibody solution by pipet completely. Add 1 mL of either 2 % HIGS washing buffer or 2% DS washing buffer.</li>
	<li>Wrap the 24 well plate in aluminium foil and wash with 3 changes of the washing buffer every 15 min with gentle mixing on a mixer at room temperature.</li>
</ol>
3. Mounting the Ear Skin on Slide
<ol>
	<li>Place the skin on the bottom of a 35 x 10 mm2 Petri dish. Carefully remove the hairs, adipose tissues, connective tissues, dusts, and fibers from the inside of the skin using fine curved forceps under stereomicroscope with low illumination to minimize extensive photo bleaching. Keep the skin wet with PBS.</li>
	<li>Transfer the skin to an adhesive microscope slide using forceps. Place the posterior and anterior skin with the inside facing up on the slide. Flatten the skin using forceps.</li>
	<li>Mount the skin in a liquid anti-fade mounting medium to avoid photobleaching and preserve fluorescent signals. Make sure that no air bubbles are on or around the skin.</li>
	<li>Cover with coverslip on the skin samples carefully and store the mounted skin sample slides in the dark overnight at room temperature to allow the mounting media get firm. Seal the coverslip to the slide with nail polish and store it at 4 &#176;C for long-term storage.</li>
</ol>
4. Confocal Microscopy
<ol>
	<li>Set up appropriate lasers for fluorophores. A confocal microscope with three laser sources (Argon 488 nm, DPSS 561 nm and HeNe 633 nm) is used in this experiment.</li>
	<li>Use the sequential scan tool, which simultaneously excites triple-stained samples, to avoid and reduce any overlap. 
	NOTE: Images will be taken in a sequential manner using the sequential scan mode.</li>
	<li>Image under a 10X objective. Use the tile scan tool to capture the whole ear skin. Set Z-stack and make sure that the z-position covers the entire thickness of ear skin.</li>
</ol>

<ol>
	<li>Mukouyama, Y. S., Shin, D., Britsch, S., Taniguchi, M., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+nerves+determine+the+pattern+of+arterial+differentiation+and+blood+vessel+branching+in+the+skin.">Sensory nerves determine the pattern of arterial differentiation and blood vessel branching in the skin.</a> Cell. 109, 693-705 (2002).</li><li>Mukouyama, Y. S., James, J., Nam, J., Uchida, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+confocal+microscopy+for+vascular+branching+morphogenesis.">Whole-mount confocal microscopy for vascular branching morphogenesis.</a> Methods Mol Biol. 843, 69-78 (2012).</li><li>Li, W., Mukouyama, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+immunohistochemical+analysis+for+embryonic+limb+skin+vasculature:+a+model+system+to+study+vascular+branching+morphogenesis+in+embryo.">Whole-mount immunohistochemical analysis for embryonic limb skin vasculature: a model system to study vascular branching morphogenesis in embryo.</a> J Vis Exp. , (2011).</li><li>Yamazaki, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Myeloid+Progenitors+Differentiate+into+Pericytes+through+TGF-beta+Signaling+in+Developing+Skin+Vasculature.">Tissue Myeloid Progenitors Differentiate into Pericytes through TGF-beta Signaling in Developing Skin Vasculature.</a> Cell Rep. 18, 2991-3004 (2017).</li><li>Mukouyama, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vessel-dependent+recruitment+of+sympathetic+axons:+looking+for+innervation+in+all+the+right+places.">Vessel-dependent recruitment of sympathetic axons: looking for innervation in all the right places.</a> J Clin Invest. 124, 2855-2857 (2014).</li><li>Li, W., 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=Peripheral+nerve-derived+CXCL12+and+VEGF-A+regulate+the+patterning+of+arterial+vessel+branching+in+developing+limb+skin.">Peripheral nerve-derived CXCL12 and VEGF-A regulate the patterning of arterial vessel branching in developing limb skin.</a> Dev Cell. 24, 359-371 (2013).</li><li>Chang, H., Wang, Y., Wu, 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=Flat+mount+imaging+of+mouse+skin+and+its+application+to+the+analysis+of+hair+follicle+patterning+and+sensory+axon+morphology.">Flat mount imaging of mouse skin and its application to the analysis of hair follicle patterning and sensory axon morphology.</a> J Vis Exp. , e51749 (2014).</li><li>Salz, L., Driskell, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Horizontal+Whole+Mount:+A+Novel+Processing+and+Imaging+Protocol+for+Thick,+Three-dimensional+Tissue+Cross-sections+of+Skin.">Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin.</a> J Vis Exp. , (2017).</li><li>Liakath-Ali, K., 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=Novel+skin+phenotypes+revealed+by+a+genome-wide+mouse+reverse+genetic+screen.">Novel skin phenotypes revealed by a genome-wide mouse reverse genetic screen.</a> Nat Commun. 5, 3540 (2014).</li><li>Gunawan, M., 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+methyltransferase+Ezh2+controls+cell+adhesion+and+migration+through+direct+methylation+of+the+extranuclear+regulatory+protein+talin.">The methyltransferase Ezh2 controls cell adhesion and migration through direct methylation of the extranuclear regulatory protein talin.</a> Nat Immunol. 16, 505-516 (2015).</li><li>Avci, P., 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=Animal+models+of+skin+disease+for+drug+discovery.">Animal models of skin disease for drug discovery.</a> Expert Opin Drug Dis. 8, 331-355 (2013).</li><li>Jin, H., He, R., Oyoshi, M., Geha, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+atopic+dermatitis.">Animal models of atopic dermatitis.</a> J Invest Dermatol. 129, 31-40 (2009).</li><li>Wagner, E. F., Schonthaler, H. B., Guinea-Viniegra, J., Tschachler, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psoriasis:+what+we+have+learned+from+mouse+models.">Psoriasis: what we have learned from mouse models.</a> Nat Rev Rheumatol. 6, 704-714 (2010).</li><li>Nunan, R., Harding, K. G., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+challenges+of+chronic+wounds:+searching+for+an+optimal+animal+model+to+recapitulate+their+complexity.">Clinical challenges of chronic wounds: searching for an optimal animal model to recapitulate their complexity.</a> Dis Model Mech. 7, 1205-1213 (2014).</li><li>O'Brien, P. D., Sakowski, S. A., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+diabetic+neuropathy.">Mouse models of diabetic neuropathy.</a> ILAR J. 54, 259-272 (2014).</li><li>Yamazaki, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-Mount+Adult+Ear+Skin+Imaging+Reveals+Defective+Neuro-Vascular++Branching+Morphogenesis+in+Obese+and+Type+2+Diabetic+Mouse+Models.">Whole-Mount Adult Ear Skin Imaging Reveals Defective Neuro-Vascular Branching Morphogenesis in Obese and Type 2 Diabetic Mouse Models.</a> Sci Rep. 8, (2018).</li></ol>

The authors have nothing to disclose.

This protocol describes the whole-mount immunonohistochemical imaging of adult ear skin for the analysis of neuro-vascular structures and immune cell distribution. We believe this method has numerous experimental advantages for researchers to study branching morphogenesis and the patterning of peripheral nerves and blood vessels, as well as three-dimensional distribution of skin components including immune cells and hair follicles. The results of the imaging can be quantified using imaging softwares for further quantitative analysis.
Proper preparation of the ear skin is critical for the success of this protocol. First, the ear skin should be carefully dissected soon after euthanizing the mouse. The posterior part of the ear skin should be peeled away from the cartilage. Then, the cartilage should be peeled off from anterior skin before staining. Second, connective tissues, adipose tissues, and hairs should be removed gently and thoroughly before mounting. Due to the existence of peripheral nerves on the surface of the skin, careful removal is required to avoid damaging the nerves. Third, the ear skin should be unfolded with the removal of some thick tissues from the ear skin. Finally, the ear skin should be flat-mounted without air bubbles.
One limitation of this protocol is that ear-tagged ear skin is not appropriate for the analysis as ear tag causes a hole or a scar. Therefore, identification of mice by different methods other than ear tag such as labeling on the tail is necessary in case there are multiple mice to analyze.
The entire ear skin can be scanned by confocal microscopy with a tile scan tool, although a previous reports demonstrated that a region of interest can be imaged with a high resolution10. Interestingly, the whole-mount imaging of the entire ear skin reveals distinct branching morphogenesis and patterning of peripheral nerves and blood vessels between the posterior and anterior skin (Figure 1): the posterior skin has large-diameter nerve bundles (20&#8211;50 &#181;m) aligned with remodeled large-diameter blood vessels covered with &#945;SMA+ VSMCs (20&#8211;60 &#181;m), while the anterior skin has smaller-diameter nerve bundles (&#60;20 &#181;m) aligned with smaller-diameter but remodeled blood vessels covered with &#945;SMA+ VSMCs (&#60;20 &#181;m).
There are a remarkable number of mouse models11 to elucidate the mechanism of human dermatological diseases such as atopic dermatitis12, psoriasis13, wound healing14, and diabetic neuropathy15. We have applied this protocol to the ear skin of diet-induced obesity mice and type 2 diabetic mice to study diabetic neuropathy16. This protocol can be applied from juvenile to adult mouse skin in various pathological conditions.

Skin is comprised of three layers: the epidermis, dermis and hypodermis. It has been used as a model system to study stem cell maintenance, differentiation, and morphogenesis in development as well as the regeneration, tumorigenesis, and inflammation in adult. Skin is richly vascularized and innervated such that the development of the peripheral nervous system and vascular system is well-coordinated.
We have previously demonstrated a whole-mount embryonic skin imaging technique with multiple labeling to study intact peripheral nerves and blood vessels including their cellular components1,2,3,4: sensory axons, sympathetic axons, Schwann cells in nerves, endothelial cells, pericytes, and vascular smooth muscle cells (VSMCs) in blood vessels. During the angiogenesis, a primary capillary network undergoes intensive vascular remodeling and develops into a hierarchical vascular branching network. In the developing dermis/hypodermis, arteries branch alongside peripheral sensory nerves and veins then form adjacent to the arteries. After the hierarchical vascular network is thoroughly covered with VSMCs, sympathetic nerves extend along and innervate large-diameter blood vessels1,5,6. Despite the significance in the close association between the developing nervous and vascular systems, a major question has been to address what happens to the neuro-vascular networks in various pathological situations in adults. A three-dimensional high-resolution imaging is necessary to appreciate the pathogenesis, along with anatomically recognizable branching morphogenesis and patterning.
Neuronal and vascular morphogenesis in adult mouse skin is commonly analyzed by tissue section staining. Other studies have used whole-mount imaging of the skin to visualize the peripheral nerves and blood vessels, in addition to the hair follicles, sebaceous glands, and arrector pili muscles7,8,9. However, the thickness of adult skin has made it difficult to analyze the skin over its entire depth.
In the present study, we developed a novel high-resolution whole-mount imaging of adult ear skin to overcome these challenges. Ear skin is readily accessible for dissection and subsequent whole-mount imaging of the skin over its entire depth. Thus, it is a straightforward and highly reproducible method that can be applied to compare three-dimensional architecture of peripheral nervous and vascular systems in the skin, with comprehensive quantification measurements. We demonstrated that the alignment of peripheral sensory and sympathetic nerves with large-diameter blood vessels is preserved in the adult skin. The goal of this protocol is to visualize branching morphogenesis, and the patterning of peripheral nerves and blood vessels, as well as the immune cell distribution at a cellular level in adult mouse models in various conditions such as inflammation and regeneration.

<table><tbody><tr><td>10 x Phosphate Buffered Saline</td><td>KD Medical</td><td>RGE-3210</td><td>PBS, without Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution</td><td>Gibco</td><td>14025-092</td><td>HBSS, with Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>16% Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td>PFA, fixative, diluted in PBS</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X100</td><td>Detergent</td></tr><tr><td>Normal goat serum</td><td>Gibco</td><td>16210064</td><td>Component of blocking/washing buffer</td></tr><tr><td>Normal donkey serum</td><td>Jackson Immuno research</td><td>017-000-121</td><td>Component of blocking/washing buffer</td></tr><tr><td>Curved fine tweezers</td><td>Dumont</td><td>RS-5047</td><td></td></tr><tr><td>Curved tweezers</td><td>Integra Miltex Vantage</td><td>V918-782, V918-784</td><td></td></tr><tr><td>Filter Unit 0.45 mm</td><td>Thermo Scientific</td><td>157-0045</td><td>For filtration</td></tr><tr><td>1 mL syringe</td><td>Coviden</td><td>8881501400</td><td>For filtration</td></tr><tr><td>Syringe filter Unit 0.22 mm</td><td>Millex-GV</td><td>SLGVR04NL</td><td>For filtration</td></tr><tr><td>ProLong Gold</td><td>Thermo Scientific</td><td>P36934</td><td>Anti-fade mounting medium</td></tr><tr><td>Nail Polish</td><td>Electron Microscopy Sciences</td><td>72180</td><td>For sealing</td></tr><tr><td>Dissecting microscope</td><td>Leica</td><td>MZ95</td><td></td></tr><tr><td>Confocal microscope</td><td>Leica</td><td>TCS SP5</td><td></td></tr><tr><td>Photoshop CC 2017</td><td>Adobe</td><td></td><td>Graphics editor software</td></tr><tr><td>Illustrator CC 2017</td><td>Adobe</td><td></td><td>Graphics editor software</td></tr><tr><td>Image J</td><td>NIH</td><td></td><td>Image processing software</td></tr><tr><td>Anti-PECAM-1 (CD31) antibody</td><td>Millipore</td><td>MAB1398Z</td><td>Hamster IgG, vascular endothelial cell marker, 1:300</td></tr><tr><td>Anti-PECAM-1 (CD31) antibody</td><td>BD Pharmingen</td><td>553369</td><td>Rat IgG2a kappa, vascular endothelial cell marker, 1:300</td></tr><tr><td>Anti-&amp;#945;SMA antibody conjugated with cy-3</td><td>Sigma</td><td>C6198</td><td>Mouse IgG2a, vascular smooth muscle cell marker, 1:500</td></tr><tr><td>Anti-EphB1 antibody</td><td>Santa Cruz</td><td>sc-9319</td><td>Goat polyclonal, venous endothelial cell marker, 1:100</td></tr><tr><td>Anti-neuron-specific Class III &amp;#946;-tubulin (Tuj1)</td><td>Abcam</td><td>AB18207</td><td>Tuj1, Rabbit polyclonal IgG, pan-axonal marker, 1:500</td></tr><tr><td>Anti-Tuj1 antibody</td><td>Covance</td><td>MMS-435P</td><td>Mouse IgG2a, pan-axonal marker, 1:500</td></tr><tr><td>Anti-MBP antibody</td><td>Abcam</td><td>AB40390</td><td>Rabbit polyclonal IgG, myelination marker, 1:200</td></tr><tr><td>Anti-Tyrosine Hydroxylase antibody</td><td>Chemicon</td><td>AB152</td><td>Rabbit polyclonal, sympathetic neuron marker, 1:500</td></tr><tr><td>Anti-Peripherin antibody</td><td>Chemicon</td><td>AB1530</td><td>Rabbit polyclonal, peripheral neuron marker, 1:1000</td></tr><tr><td>Anti-CD11b antibody</td><td>Bio-Rad</td><td>MCA74G</td><td>Rat IgG2b, inflammatory cell marker (macrophages), 1:50</td></tr><tr><td>Anti-CD45 antibody</td><td>Thermo Fisher Scientific</td><td>14-0451-85</td><td>Rat IgG2b kappa, pan-hematopoietic cell marker, 1:500</td></tr><tr><td>Anti-CD3 antibody</td><td>Bio-Rad</td><td>MCA1477T</td><td>Rat IgG1, immune cell marker, 1:100</td></tr><tr><td>Anti-CD45R (B220) antibody</td><td>Thermo Fisher Scientific</td><td>14-0452</td><td>Rat IgG2a kappa, inflammatory cell marker, 1:200</td></tr><tr><td>Anti-GFP antibody</td><td>Thermo Fisher Scientific</td><td>A11122</td><td>Rabbit polyclonal, 1:300</td></tr><tr><td>Anti-GFP antibody</td><td>Abcam</td><td>Ab13970</td><td>Chicken polyclonal, 1:500</td></tr><tr><td>Anti-&amp;#946;-gal antibody</td><td>Cappel</td><td>55976</td><td>Rabbit polyclonal, 1:5000</td></tr><tr><td>Anti-RFP antibody</td><td>Abcam</td><td>Ab62341</td><td>Rabbit polyclonal, 1:300</td></tr><tr><td>Goat anti-rabbit IgG (H+L) Alexa 488</td><td>Thermo Fisher Scientific</td><td>A11034</td><td>Rabbit polyclonal secondary antibody, 1:250</td></tr><tr><td>Goat anti-hamster IgG (H+L) Alexa 647</td><td>Jackson Immuno research</td><td>127-605-160</td><td>Hamster polyclonal secondary antibody, 1:250</td></tr><tr><td>Goat anti-rat IgG (H+L) Alexa 594</td><td>Jackson Immuno research</td><td>112-585-167</td><td>Rat polyclonal secondary antibody, 1:250</td></tr><tr><td>Goat anti-mouse IgG2a Alexa 633</td><td>Thermo Fisher Scientific</td><td>A21136</td><td>Mouse IgG2a secondary antibody, 1:250</td></tr></tbody></table>

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.

Adult mouse posterior ear skin (Figure 1A) and anterior ear skin (Figure 1B) were immunostained with antibodies to &#945;SMA (red), Tuj1 (green), and PECAM-1 (blue). Posterior skin was immunostained to study neuro-immune distribution using antibodies to CD11b (red) and MBP (green), together with Tuj1 (blue) (Figure 2A). Distribution of CD11b+ inflammatory cells, including macrophages was detected at a single cellular resolution (Figure 2B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57406/57406fig1.jpg" /> 
Figure 1: Alignment ofperipheral nerves and blood vessels in adult ear skin. Whole-mount triple immunofluorescence confocal microscopy of posterior and anterior ear skin with antibodies to &#945;SMA (red), Tuj1 (green), and PECAM-1 (blue) is shown. (A) VSMC-covered large-diameter blood vessels align with peripheral nerves in the posterior ear skin. (B) Smaller-diameter blood vessels covered with VSMCs align with smaller-diameter peripheral nerve bundles in the anterior ear skin. Scale bar = 1 mm <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57406/57406fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57406/57406fig2.jpg" /> 
Figure 2: Myelination of peripheral nerves and CD11b+ myeloid cell distribution in adult ear skin. Whole-mount triple immunofluorescence confocal microscopy of posterior ear skin with antibodies to CD11b (red) and MBP (green), together with Tuj1 (blue), is shown. (A) Medium-to-large diameter peripheral nerves are myelinated. (B) Close-up image in (A). CD11b+ inflammatory cells distribute evenly in the posterior ear skin. Scale bar = 1 mm (A), 100 &#181;m (B). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57406/57406fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution at JoVE.com

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

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 ...

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Nanyang Technological University

Research

JoVE Journal

Developmental Biology

13.0K Views.  National Institutes of Health. Hier beschrijven we een hoge resolutie geheel-mount beeldvorming methode in de hele volwassen muis oor huid, waarmee we kunnen visualiseren vertakkende genuitdrukking en patronen van perifere zenuwen en bloedvaten, evenals immuun cel distributie.

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

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.

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 ...

Neuroscience

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Immunology and Infection

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 ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Biology

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 ...

Cryosectioning and Immunostaining Mouse Inner Ear Tissue: From Embryonic to Adult Stages

This protocol details general histology methods for preparing inner ear samples from embryonic, neonatal, and adult mice. The purpose of this protocol is to provide a straightforward and standardized method for inner ear tissue processing for researchers, possibly new to the field, interested in working with the mouse cochlea. Included here are protocols for dissection, fixation, embedding, cryosectioning, and immunostaining. Section immunostaining is one of the best methods for examining individual cells within the context of the entire cochlea.
Key steps include dissecting the inner ear away from the skull, fixing the tissue using 4% paraformaldehyde, embedding with Optimal Cutting Temperature compound, cryosectioning, and immunostaining with antibodies targeting specific proteins expressed by the cochlea.
Included in our methods are special considerations made for the cochlea given its 
shape and structure.&#160;Reasonably good focus and dissection skills are needed, as tissue damage can affect the quality of immunostaining and consistency among samples. However, with the procedures we outline, most users can be trained in a few weeks to make beautiful preparations. Overall, this protocol offers a valuable way to promote research to understand auditory development, function, and disease in mouse models.

This protocol, intended for novice users of mouse inner ear tissue, comprehensively details steps for processing mouse inner ears at different developmental stages for section immunostaining.

This protocol details general histology methods for preparing inner ear samples from embryonic, neonatal, and adult mice. The purpose of this protocol is ...

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent re-oxygenation of the tissues (reperfusion). In the skin, ischemia-reperfusion (IR) is the main contributing factor to the pathophysiology of pressure ulcers. While the cascade of events leading up to the inflammatory response has been well studied, the spatial and temporal responses of the different subsets of immune cells to an IR injury are not well understood. Existing models of IR using the clamping technique on the skin flank are highly invasive and unsuitable for studying immune responses to injury, while similar non-invasive magnet clamping studies in the skin flank are less-than-ideal for intravital imaging studies. In this protocol, we describe a robust model of non-invasive IR developed on mouse ear skin, where we aim to visualize in real-time the cellular response of immune cells after reperfusion via multiphoton intravital imaging (MP-IVM).

This protocol describes the induction of an ischemia-reperfusion (IR) model on mouse ear skin using magnet clamping. Using a custom-built intravital imaging model, we study in vivo inflammatory responses post-reperfusion. The rationale behind the development of this technique is to extend the understanding of how leukocytes respond to skin IR injury.

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent ...

Medicine

Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes the atrioventricular node (AVN), to allow excitation and contraction of the whole heart. Any dysfunction of either SAN or AVN results in arrhythmias, indicating their fundamental role in electrophysiology and arrhythmogenesis. Mouse models are widely used in arrhythmia research, but the specific investigation of SAN and AVN remains challenging.
The SAN is located at the junction of the crista terminalis with the superior vena cava and AVN is located at the apex of the triangle of Koch, formed by the orifice of the coronary sinus, the tricuspid annulus, and the tendon of Todaro. However, due to the small size, visualization by conventional histology remains challenging and it does not allow the study of SAN and AVN within their 3D environment.
Here we describe a whole-mount immunofluorescence approach that allows the local visualization of labelled mouse SAN and AVN. Whole-mount immunofluorescence staining is intended for smaller sections of tissue without the need for manual sectioning. To this purpose, the mouse heart is dissected, with unwanted tissue removed, followed by fixation, permeabilization and blocking. Cells of the conduction system within SAN and AVN are then stained with an anti-HCN4 antibody. Confocal laser scanning microscopy and image processing allow differentiation between nodal cells and working cardiomyocytes, and to clearly localize SAN and AVN. Furthermore, additional antibodies can be combined to label other cell types as well, such as nerve fibers.
Compared to conventional immunohistology, whole-mount immunofluorescence staining preserves the anatomical integrity of the cardiac conduction system, thus allowing the investigation of AVN; especially so into their anatomy and interactions with the surrounding working myocardium and non-myocyte cells.

We provide a step-by-step protocol for whole-mount immunofluorescence staining of the sinoatrial node (SAN) and atrioventricular node (AVN) in murine hearts.

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes ...

Stria Vascularis Microdissection for Single-Nucleus Sequencing or Immunostaining

Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining

Endocochlear potential, which is generated by the stria vascularis, is essential to maintain an environment conducive to appropriate hair cell mechanotransduction and ultimately hearing. Pathologies of the stria vascularis can result in a decreased hearing. Dissection of the adult stria vascularis allows for focused single-nucleus capture and subsequent single-nucleus sequencing and immunostaining. These techniques are used to study stria vascularis pathophysiology at the single-cell level. 
Single-nucleus sequencing can be used in the setting of transcriptional analysis of the stria vascularis. Meanwhile, immunostaining continues to be useful in identifying specific populations of cells. Both methods require proper stria vascularis dissection as a prerequisite, which can prove to be technically challenging.

Author Spotlight: Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining  

The stria vascularis is vital to the generation of endocochlear potential. Here, we present the dissection of the adult mouse stria vascularis for single-nucleus sequencing or immunostaining.

Endocochlear potential, which is generated by the stria vascularis, is essential to maintain an environment conducive to appropriate hair cell ...

Intravital Imaging Using an Inverted Microscope to Track Skin Cell Dynamics

Streamlined Intravital Imaging Approach for Long-Term Monitoring of Epithelial Tissue Dynamics on an Inverted Confocal Microscope

Understanding normal and aberrant in vivo cell behaviors is necessary to develop clinical interventions to thwart disease initiation and progression. It is therefore critical to optimize imaging approaches that facilitate the observation of cell dynamics in situ, where tissue structure and composition remain unperturbed. The epidermis is the body&#39;s outermost barrier, as well as the source of the most prevalent human cancers, namely cutaneous skin carcinomas. The accessibility of skin tissue presents a unique opportunity to monitor epithelial and dermal cell behaviors in intact animals using noninvasive intravital microscopy. Nevertheless, this sophisticated imaging approach has primarily been achieved using upright multiphoton microscopes, which represent a significant barrier for entry for most investigators. This study presents a custom-designed, 3D-printed microscope stage insert suitable for use with inverted confocal microscopes, streamlining the long-term intravital imaging of ear skin in live transgenic mice. We believe this versatile invention, which may be customized to fit the inverted microscope brand and model of choice and adapted to image additional organ systems, will prove invaluable to the greater scientific research community by significantly enhancing the accessibility of intravital microscopy. This technological advancement is critical for bolstering our understanding of live cell dynamics in normal and disease contexts.

Author Spotlight: Developing a Tool for Using Inverted Confocal Microscopes for In Vivo Intravital Imaging 

The protocol presents a new tool to simplify intravital imaging using inverted confocal microscopy.

Understanding normal and aberrant in vivo cell behaviors is necessary to develop clinical interventions to thwart disease initiation and progression. It ...

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

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