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
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.
3. Mounting the Ear Skin on Slide
4. Confocal Microscopy
Adult mouse posterior ear skin (Figure 1A) and anterior ear skin (Figure 1B) were immunostained with antibodies to αSMA (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).
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 αSMA (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 Please click here to view a larger version of this figure.
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 µm (B). Please click here to view a larger version of this figure.
10 x Phosphate Buffered Saline | KD Medical | RGE-3210 | PBS, without Ca2+/Mg2+ |
Hank’s Balanced Salt Solution | Gibco | 14025-092 | HBSS, with Ca2+/Mg2+ |
16% Paraformaldehyde | Electron Microscopy Sciences | 15710 | PFA, fixative, diluted in PBS |
Triton X-100 | Sigma | X100 | Detergent |
Normal goat serum | Gibco | 16210064 | Component of blocking/washing buffer |
Normal donkey serum | Jackson Immuno research | 017-000-121 | Component of blocking/washing buffer |
Curved fine tweezers | Dumont | RS-5047 | |
Curved tweezers | Integra Miltex Vantage | V918-782, V918-784 | |
Filter Unit 0.45 mm | Thermo Scientific | 157-0045 | For filtration |
1 mL syringe | Coviden | 8881501400 | For filtration |
Syringe filter Unit 0.22 mm | Millex-GV | SLGVR04NL | For filtration |
ProLong Gold | Thermo Scientific | P36934 | Anti-fade mounting medium |
Nail Polish | Electron Microscopy Sciences | 72180 | For sealing |
Dissecting microscope | Leica | MZ95 | |
Confocal microscope | Leica | TCS SP5 | |
Photoshop CC 2017 | Adobe | Graphics editor software | |
Illustrator CC 2017 | Adobe | Graphics editor software | |
Image J | NIH | Image processing software | |
Anti-PECAM-1 (CD31) antibody | Millipore | MAB1398Z | Hamster IgG, vascular endothelial cell marker, 1:300 |
Anti-PECAM-1 (CD31) antibody | BD Pharmingen | 553369 | Rat IgG2a kappa, vascular endothelial cell marker, 1:300 |
Anti-aSMA antibody conjugated with cy-3 | Sigma | C6198 | Mouse IgG2a, vascular smooth muscle cell marker, 1:500 |
Anti-EphB1 antibody | Santa Cruz | sc-9319 | Goat polyclonal, venous endothelial cell marker, 1:100 |
Anti-neuron-specific Class III b-tubulin (Tuj1) | Abcam | AB18207 | Tuj1, Rabbit polyclonal IgG, pan-axonal marker, 1:500 |
Anti-Tuj1 antibody | Covance | MMS-435P | Mouse IgG2a, pan-axonal marker, 1:500 |
Anti-MBP antibody | Abcam | AB40390 | Rabbit polyclonal IgG, myelination marker, 1:200 |
Anti-Tyrosine Hydroxylase antibody | Chemicon | AB152 | Rabbit polyclonal, sympathetic neuron marker, 1:500 |
Anti-Peripherin antibody | Chemicon | AB1530 | Rabbit polyclonal, peripheral neuron marker, 1:1000 |
Anti-CD11b antibody | Bio-Rad | MCA74G | Rat IgG2b, inflammatory cell marker (macrophages), 1:50 |
Anti-CD45 antibody | Thermo Fisher Scientific | 14-0451-85 | Rat IgG2b kappa, pan-hematopoietic cell marker, 1:500 |
Anti-CD3 antibody | Bio-Rad | MCA1477T | Rat IgG1, immune cell marker, 1:100 |
Anti-CD45R (B220) antibody | Thermo Fisher Scientific | 14-0452 | Rat IgG2a kappa, inflammatory cell marker, 1:200 |
Anti-GFP antibody | Thermo Fisher Scientific | A11122 | Rabbit polyclonal, 1:300 |
Anti-GFP antibody | Abcam | Ab13970 | Chicken polyclonal, 1:500 |
Anti-b-gal antibody | Cappel | 55976 | Rabbit polyclonal, 1:5000 |
Anti-RFP antibody | Abcam | Ab62341 | Rabbit polyclonal, 1:300 |
Goat anti-rabbit IgG (H+L) Alexa 488 | Thermo Fisher Scientific | A11034 | Rabbit polyclonal secondary antibody, 1:250 |
Goat anti-hamster IgG (H+L) Alexa 647 | Jackson Immuno research | 127-605-160 | Hamster polyclonal secondary antibody, 1:250 |
Goat anti-rat IgG (H+L) Alexa 594 | Jackson Immuno research | 112-585-167 | Rat polyclonal secondary antibody, 1:250 |
Goat anti-mouse IgG2a Alexa 633 | Thermo Fisher Scientific | A21136 | Mouse IgG2a secondary antibody, 1:250 |
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 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 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.