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
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

Agradecemos a K. Gill para a gestão do laboratório e suporte técnico, J. Hawkins e os funcionários dos institutos nacionais de saúde (NIH) facilidade do edifício 50 animais de assistência com cuidados de rato e R. Reed e F. Bernardo para assistência administrativa. Obrigado também a Motegi S. e M. Udey por compartilhar o seu protocolo de dissecação de pele orelha, s. Burns para ajuda editorial e membros do laboratório de células-tronco e biologia Neuro-Vascular para ajuda técnica e discussão pensativo. T. Yamazaki foi apoiado pela sociedade de Japão para a promoção da ciência (JSPS) NIH-KAITOKU. Este trabalho foi financiado pelo programa de pesquisa Intramural de nacional do coração, pulmão e Instituto de sangue (HL005702-11 para Y.M.)

Todos os experimentos nesta seção foram realizados sob aprovação do nacional do coração, pulmão e cuidado do Animal de sangue Institute (NHLBI) e Comité de uso.
1. coleção de pele de orelha de rato adulto
<ol><li>Eutanásia em ratos adultos pela exposição de dióxido de carbono (CO2) em uma câmara fechada e em seguida, confirme a eutanásia por deslocamento cervical. Nota: O experimento segue a diretriz do National Institutes of Health (NIH) para o método de eutan...

<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>

Este protocolo descreve a imagem de toda a montagem immunonohistochemical de pele de orelha do adulto para a análise das estruturas neuro-vasculares e distribuição de células imunes. Acreditamos que este método tem várias vantagens experimentais aos pesquisadores estudar ramificação morfogênese e a padronização de nervos periféricos e os vasos sanguíneos, bem como a distribuição tridimensional dos componentes da pele, incluindo o cabelo e células do sistema imunológico folículos. Os resultados da imagem...

Pele é composta por três camadas: epiderme, derme e hipoderme. Ela tem sido usada como um sistema modelo para estudar a manutenção de células-tronco, diferenciação e morfogênese em desenvolvimento, bem como a regeneração tumorigênese e inflamação em adultos. Pele é ricamente vascularizada e inervada tal que o desenvolvimento do sistema vascular e sistema nervoso periférico é bem coordenado.
Nós demonstramos anteriormente uma técnica de imagem de montagem em toda a pele embrionária com múltiplos rotulagem para estudar os nervos periféricos intactos e os vasos sanguíneos, incluindo seus componentes celulares1,2,3, 4: axônios sensoriais, simpáticos axônios, células em nervos Schwann, células endoteliais, pericitos e células de músculo liso vascular (VSMCs) nos vasos sanguíneos. Durante a angiogênese, uma rede capilar primária sofre remodelação vascular intensivo e se desenvolve em uma rede hierárquica de ramificação vascular. Na derme/hipoderme em desenvolvimento, as artérias ramificam ao lado de veias e nervos sensoriais periféricos dão forma então adjacentes às artérias. Depois que a rede vascular hierárquica é completamente coberta com VSMCs, nervos simpáticos se estendem ao longo e inervam os vasos sanguíneos de grande diâmetro1,5,6. Apesar da importância da associação estreita entre os sistemas nervosos e vasculares em desenvolvimento, uma grande questão tem sido a abordar o que acontece com as redes neuro-vascular em diversas situações patológicas em adultos. Uma imagem tridimensional de alta resolução é necessária apreciar a patogênese, juntamente com a morfogênese ramificação anatomicamente reconhecível e padronização.
Morfogênese neuronal e vascular em pele de rato adulto é comumente analisada pela coloração de secção de tecido. Outros estudos utilizaram imagens de toda a montagem da pele para visualizar os nervos periféricos e dos vasos sanguíneos, além dos folículos pilosos, glândulas sebáceas e arrector pili músculos7,8,9. No entanto, a espessura da pele adulta tornou difícil analisar a pele ao longo de toda a sua profundidade.
No presente estudo, nós desenvolvemos uma romance de alta resolução de imagem toda a montagem da pele da orelha do adulto para superar estes desafios. Pele da orelha é prontamente acessível para dissecção e subsequente de imagem de toda a montagem da pele ao longo de toda a sua profundidade. Assim, é um método simples e altamente reprodutível que pode ser aplicado para comparar a arquitetura tridimensional dos sistemas nervoso e vasculares periféricas na pele, com medidas de quantificação abrangente. Demonstrámos que o alinhamento dos nervos periféricos sensoriais e simpáticos com os vasos sanguíneos de grande diâmetro é preservado na pele adulta. O objetivo do presente protocolo é Visualizar ramificação morfogênese e a padronização dos nervos periféricos e os vasos sanguíneos, bem como a distribuição de células imunes a um nível celular em modelos do rato adulto em várias condições, tais como inflamação e regeneração.

<table>
	<tbody>
		<tr>
			<td>10 x Phosphate Buffered Saline</td>
			<td>KD Medical</td>
			<td>RGE-3210</td>
			<td>PBS, without Ca2+/Mg2+</td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution</td>
			<td>Gibco</td>
			<td>14025-092</td>
			<td>HBSS, with Ca2+/Mg2+</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-aSMA 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 b-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-b-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

Aqui, apresentamos um protocolo de uma pele de orelha do adulto de toda a montagem de imagem técnica para estudo abrangente tridimensional neuro-vascular ramificação morfogênese e padronização, bem como a distribuição de células imunes a um nível celular. A análise das estruturas anatômicas nervos e dos vasos sanguíneos periféricas em tecidos adultos fornece alguns insights sobre a compreensão da fiação neuro-vascular funcional e degeneração neuro-vascular em condições patológicas como a cicatrização de feridas. Como um sistema modelo altamente informativa, focamos nossos estudos na pele da orelha do adulto, que é facilmente acessível para dissecação. Nosso protocolo simples e reprodutível fornece uma descrição exata dos componentes celulares da pele inteira, tais como os nervos periféricos (axônios sensoriais, simpáticos axônios e células de Schwann), vasos sanguíneos (células endoteliais e células de músculo liso vascular ) e células inflamatórias. Acreditamos que este protocolo irá pavimentar o caminho para investigar as anormalidades morfológicas nos nervos periféricos e os vasos sanguíneos, bem como a inflamação da pele da orelha do adulto sob diferentes condições patológicas.

Pele de rato adulto orelha posterior (figura 1A) e pele de orelha anterior (figura 1B) foram immunostained com anticorpos αSMA (vermelho), Tuj1 (verde) e PECAM-1 (azul). Pele posterior foi immunostained para estudar a distribuição de neuro-imune usando anticorpos anti-CD11b (vermelho) e MBP (verde), juntamente com Tuj1 (azul) (Figura 2A). Distribuição de CD11b+ células inflamatórias...

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

Aqui, descrevemos um método de imagem de toda a montagem de alta resolução da pele de orelha inteira rato adulto, que nos permite visualizar ramificação morfogênese e padronização de nervos periféricos e os vasos sanguíneos, bem como a distribuição de células imunes.

Toda a montagem em microscopia Confocal para pele da orelha do adulto: um sistema de modelo para estudar a morfogênese Neuro-vasculares de ramificação e distribuição de células imunes

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

The overall goal of this procedure is to visualize branching morphogenesis and patterning of peripheral nerves and blood vessels, as well as immune cell distribution. This method can help answer key questions in the fields of neurobiology, vascular biology, and immunology by illuminating morphological abnormalities in peripheral nerves and blood vessels, as well as inflammation. The main strength of this technique is that we can visualize the cellular components of peripheral nerves, blood vessels, and immune cells in the skin over its entire depth and can compare three-dimensional architecture on nervous and vascular systems.This method can provide insight into neuro-vasculature structures and immune cell distribution in juvenile to adult mice under normal and pathology conditions. After euthanizing adult mice according to the text protocol, dissect the ear from the base, and place it in a 35-by-10-millimeter Petri dish containing two milliliters of HBSS. Then, use scissors to briefly trim the hairs.Carefully peel the posterior and anterior skin away from the intervening cartilage. Transfer each section of skin separately to a 24-well plate containing one milliliter of ice-cold, fresh 4%PFA in PBS per well. Then, flatten the skin in the PFA.Next, fix the posterior and anterior skin with cartilage at four degrees Celsius for one hour. Then, use one milliliter of PBS to wash the skin three times for five minutes with gentle mixing at room temperature. Transfer the skin with cartilage to the bottom of a 35-by-10-millimeter Petri dish.Use fine curved forceps to peel off the cartilage from the anterior skin. Then, cut off the base region, which is folded and has adipose and connective tissues. Using the same forceps, carefully remove the hairs, adipose tissue, and connective tissue from the inside of the posterior skin.Use PBS to keep the skin wet. To carry out whole-mount immunohistochemical staining, transfer the posterior and anterior skin to a 24-well plate containing one milliliter of 10%heat-inactivated serum blocking buffer per well. Incubate the skin with gentle mixing at room temperature for 30 minutes.Dilute the primary antibodies in the blocking buffer as described in the text protocol, and add 150 microliters of primary antibodies in blocking buffer to the well. Then, transfer the posterior and anterior skin to new wells containing primary antibodies. Incubate the skin with gentle mixing at four degrees Celsius overnight.The following day, after transferring the skin to new wells or aspirating the primary antibodies, add one milliliter of 2%serum in PBS with 0.2%Triton X-100. Wash the samples with gentle mixing at room temperature for 15 minutes. Repeat the wash two more times.In the meantime, prepare secondary antibodies in 10%blocking buffer and filter the antibody solutions through a 0.22-micrometer PVDF membrane syringe filter connected to a one-milliliter syringe. Centrifuge the solution at 13, 000 times gravity for 10 minutes to remove aggregated particles of the secondary antibodies from the blocking buffer, adding 150 microliters of secondary antibodies in blocking buffer to the new well. Transfer the skin to the well containing secondary antibodies.Wrap the 24-well plate in aluminum foil to protect it from light, and incubate the skin with gentle mixing at room temperature for one hour. Add one milliliter of 2%serum washing buffer. Then, transfer the posterior and anterior skin to the wells containing washing buffer.Wrap the plate, and wash the samples with gentle mixing at room temperature for 15 minutes. Repeat the wash two more times. Place the skin on the bottom of a 35-by-10-millimeter Petri dish.Then, under a stereo microscope with low illumination to minimize photo bleaching, use fine curved forceps to carefully remove the hairs, adipose tissue, connective tissue, dust, and fibers from the inside of the skin. Use PBS to keep the skin wet. With forceps, transfer the posterior and anterior skin, with the inside facing up, on an adhesive microscope slide.Use forceps to flatten the skin. Mount the skin in a liquid anti-fade mounting medium to avoid photo-bleaching and preserve fluorescent signals. Make sure that no air bubbles are on or around the skin.Store the stained skin sample slides in the dark overnight at room temperature to allow the mounting medium to get firm. Then, use nail polish to seal the coverslip to the slide, and store it at four degrees Celsius for long-term storage. To carry out confocal microscopy, set up appropriate lasers for fluorophores.A confocal microscope with three laser sources is used in this experiment. Image the samples under a 10X objective, and use the sequential scan tool, which simultaneously excites triple-stained samples, to avoid and reduce any overlap. In this experiment, adult mouse posterior ear skin and anterior ear skin were immunostained with antibodies to alpha-SMA, Tuj1, and PECAM-1.The posterior skin was immunostained to study neuroimmune distribution using antibodies to CD11b and MBP, together with Tuj1. Finally, as shown here, distribution of CD11b-positive inflammatory cells, including macrophages, was detected at a single-cell resolution. While attempting this procedure, it's important to remember to handle your skin with care and to gently and slowly remove the hairs, adipose tissue, and connective tissue from the inside of the ear skin before mounting.This technique will pave the way for researchers to study branching morphogenesis and patterning of peripheral nerves and blood vessels, as well as three-dimensional distribution of skin components, including immune cells in juvenile to adult mouse models. Thanks for watching. Good luck with your experiments.

Research

JoVE Journal

Developmental Biology

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Imagens ao vivo da imunológica inata e pré-neoplásicas Interações de células utilizando um / UAS Expression System Inducible Gal4 em larvas de peixe-zebra da pele

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

Biologia do Desenvolvimento

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Conversão epigenética como um método seguro e simples para obter células secretoras de insulina a partir de fibroblastos da pele de adultos

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

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

Horizontal Whole Mount: um protocolo de processamento e imagem de Novel para secções transversais grossas de tecidos tridimensionais

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

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

Análise quantitativa da imunofluorescência toda a montagem das populações do Progenitor cardíaca em embriões de rato

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Um método simplificado e eficiente para isolar os queratinócitos humanos primários do tecido da pele de adultos

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

Visualizando o nó e placa Notochordal em Gastrulating embriões de rato usando microscopia eletrônica e imunofluorescência de montagem inteira

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

Um modelo In Vitro de um sistema de perfusão paralelo-placa para estudar a aderência bacteriana para enxertar tecidos

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

Análise do desenvolvimento de câmara cardíaca durante a embriogênese de rato usando toda montagem de epifluorescência

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Um método de montagem em camadas para a microscopia confocal de lapso de tempo prolongado de embriões zebrafish inteiros

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Hibridização em Embriões de Zebrafish e Ensaio de Formação de Tubos em iPSC-CEs para estudar o papel da endoglina no desenvolvimento vascular

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...