Source: Liang, H. et al. CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations. J. Vis. Exp (2016)
This video describes the CUBIC-based visualization technique, a tissue clearing-dependent imaging technique for visual assessment of protein expression in whole-mount skin biopsies at single-cell resolution in 3D. This technically simple protocol allows the visualization of skin proteins without preparing skin sections.
All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of the Transparent Mouse Skin Tissue
NOTE: All mice used in this study were on a C57BL/6 genetic background
2. Immunofluorescence Staining
3. Imaging
Figure 1: Preparation of the Imaging Chamber. A: Consumables required for the preparation of the imaging chamber are blue tack, play dough or similar, and 2 coverslips (24 x 50 mm). B: Prepare two thin strips of blue tack (diameter approximately 2 mm x 2 cm), and two coverslips. C: Place blue tack strips on one coverslip, allowing enough space for the skin biopsy. Place the skin biopsy in a drop of CUBIC2 solution in between the two strips. D: Place the second coverslip on blue tack strips to cover the skin biopsy.
The authors have nothing to disclose.
Paraformaldehyde | Sigma-Aldrich | P6418 | |
Ethanol 96% (undenaturated) | Chem-supply | UN1170 | |
4’,6-diamidino-2-phenylindole (DAPI) |
Roche | 10236276001 | |
N,N,N’,N’-tetrakis (2-hydroxypropyl) ethylenediamine | Merck Millipore | 821940 | |
Polyethylene glycol mono-p-isooctylphenyl ether | Merck Millipore | 648462 | |
Triton X-100 | Merck Millipore | 648462 | |
Sucrose | Sigma-Aldrich | S0389 | |
Optimal Cutting Temperature (OCT) Compound |
Tissue-Tek | 4583 | |
anti-Keratin14 antibody | Covance | PRB-155P | |
anti-Ki67 antibody | Abcam | ab16667 | |
Donkey anti-rabbit Alexa 594 | Life Technologies | A21207 | |
Dimethylsulfoxide | Sigma-Aldrich | D2650 | |
Urea | Merck Millipore | 66612 | |
2,2′,2′’-nitrilotriethanol | Merck Millipore | 137002 | |
Confocal Microscope | Nikon Instruments Inc | Nikon A1 – Confocal Microscope | |
cruZer6 Face Trimmer | Braun | Braun cruZer6 Face | |
Sodium azide | Sigma-Aldrich | 438456 |