CUBIC-based Protein Expression Visualization: A Procedure for Visualizing Protein Expression in Full Thickness Mouse Skin Biopsies

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

1. Collection of mouse skin tissue.
   1. Humanely euthanize the mice by cervical dislocation.
   2. Carefully remove hairs from the relevant skin area with a trimmer taking care not to wound the skin.
   3. Wash the skin to decontaminate with 70% ethanol in Phosphate Buffered Saline (PBS).
   ....

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 appro.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Paraformaldehyde	Sigma-Aldrich	P6418
Ethanol 96% (undenaturated)	Chem-supply	UN1170
4&rsquo;,6-diamidino-2-phenylindole<br/> (DAPI)	Roche	10236276001
N,N,N&rsquo;,N&rsquo;-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<br/> (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&prime;,2&prime;&rsquo;-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