CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

Abstract

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

Three-Dimensional Visualization of Hypoxia-Induced Pulmonary Vascular Remodeling in Mice

“…Postcaval lobes of the right lung were excised and continuously immersed in the CUBIC-1 reagent at 37 °C, either for 5 days for whole-mount staining or for 1 day for fluorescent protein labeling (Figure [A]). Next, for whole-mount immunostaining, lungs were immersed in 20% (wt/vol) sucrose in phosphate-buffered saline, frozen in optimal cutting temperature compound at −80 °C overnight to increase antibody penetration, 2 thawed at room temperature, and then subjected to whole-mount immunostaining with primary antibodies diluted in 2% (v/v) Triton X-100–phosphate-buffered saline for 3 days. The fluorescent protein–labeled and –stained samples were finally immersed in CUBIC-2 reagent for 1 day before imaging by multiphoton excitation fluorescence microscopy with tile scanning.…”

“…Immediate and sequential perfusion of fixative solution and 50% (v/v) CUBIC-1 reagent were crucial for better tissue transparency and clear 3D visualization. 1 Tissue freezing and thawing in optimal cutting temperature compound 2 and the use of higher detergent concentration (2% of Triton X-100) in antibody solution 3 were needed to increase antibody penetration and signal intensities. Because we could not optimize conditions for visualizing ECs using anti-CD31 and anti-CD102 antibodies or isolectin-B4, ECs were 3D imaged using EC-specific tdTomato fluorescence.…”