CUBIC-based Protein Expression Visualization: A Procedure for Visualizing Protein Expression in Full Thickness Mouse Skin Biopsies
Published: April 30, 2023
Abstract
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.
Protocol
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 Collection of mouse skin tissue. Humanely euthanize the mice by cervical dislocation. Carefully remove hairs from the relevant skin area wi…
Representative Results
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…
Disclosures
The authors have nothing to disclose.
Materials
| 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 |
Cite This Article
CUBIC-based Protein Expression Visualization: A Procedure for Visualizing Protein Expression in Full Thickness Mouse Skin Biopsies. J. Vis. Exp. (Pending Publication), e20359, doi: (2023).