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

Liang, Huazheng; Akladios, Bassem; Canales, Cesar P; Francis, Richard; Hardeman, Edna C.; Beverdam, Annemiek

doi:10.3791/54401

Cited by 7 publications

(7 citation statements)

References 13 publications

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“…Optic nerves were cleared using the CUBIC protocol ( Susaki et al, 2014 ). Briefly, after washing the nerves in PBS, they were kept in scale 1 solution ( Susaki et al, 2015 ; Liang et al, 2016 ) at 37°C for 4 days. Nerves were mounted in the same solution for imaging.…”

Section: Methodsmentioning

confidence: 99%

Neuroprotection and Axonal Regeneration Induced by Bone Marrow Mesenchymal Stromal Cells Depend on the Type of Transplant

Norte-Muñoz

Lucas‐Ruiz

Gallego‐Ortega

et al. 2021

Front. Cell Dev. Biol.

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Mesenchymal stromal cell (MSC) therapy to treat neurodegenerative diseases has not been as successful as expected in some preclinical studies. Because preclinical research is so diverse, it is difficult to know whether the therapeutic outcome is due to the cell type, the type of transplant or the model of disease. Our aim here was to analyze the effect of the type of transplant on neuroprotection and axonal regeneration, so we tested MSCs from the same niche in the same model of neurodegeneration in the three transplantation settings: xenogeneic, syngeneic and allogeneic. For this, bone marrow mesenchymal stromal cells (BM-MSCs) isolated from healthy human volunteers or C57/BL6 mice were injected into the vitreous body of C57/BL6 mice (xenograft and syngraft) or BALB/c mice (allograft) right after optic nerve axotomy. As controls, vehicle matched groups were done. Retinal anatomy and function were analyzed in vivo by optical coherence tomography and electroretinogram, respectively. Survival of vision forming (Brn3a+) and non-vision forming (melanopsin+) retinal ganglion cells (RGCs) was assessed at 3, 5 and 90 days after the lesion. Regenerative axons were visualized by cholera toxin β anterograde transport. Our data show that grafted BM-MSCs did not integrate in the retina but formed a mesh on top of the ganglion cell layer. The xenotransplant caused retinal edema, detachment and folding, and a significant decrease of functionality compared to the murine transplants. RGC survival and axonal regeneration were significantly higher in the syngrafted retinas than in the other two groups or vehicle controls. Melanopsin+RGCs, but not Brn3a+RGCs, were also neuroprotected by the xenograft. In conclusion, the type of transplant has an impact on the therapeutic effect of BM-MSCs affecting not only neuronal survival but also the host tissue response. Our data indicate that syngrafts may be more beneficial than allografts and, interestingly, that the type of neuron that is rescued also plays a significant role in the successfulness of the cell therapy.

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Section: Methodsmentioning

confidence: 99%

Neuroprotection and Axonal Regeneration Induced by Bone Marrow Mesenchymal Stromal Cells Depend on the Type of Transplant

Norte-Muñoz

Lucas‐Ruiz

Gallego‐Ortega

et al. 2021

Front. Cell Dev. Biol.

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“…CUBIC was used in the study of mammary glands and their differentiation as well as cells fate in mice mammary gland ducts (74). The CUBIC protocol was used to investigate skin stem cells in full thickness skin biopsies (191). HSCs were also imaged in the spleen, brain and thymocytes after clearing them cleared with CUBIC method (192).…”

Section: Cubic For 3d Imaging Of Stem Cellsmentioning

confidence: 99%

Three-Dimensional Imaging in Stem Cell-Based Researches

et al. 2021

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Stem cells have an important role in regenerative therapies, developmental biology studies and drug screening. Basic and translational research in stem cell technology needs more detailed imaging techniques. The possibility of cell-based therapeutic strategies has been validated in the stem cell field over recent years, a more detailed characterization of the properties of stem cells is needed for connectomics of large assemblies and structural analyses of these cells. The aim of stem cell imaging is the characterization of differentiation state, cellular function, purity and cell location. Recent progress in stem cell imaging field has included ultrasound-based technique to study living stem cells and florescence microscopy-based technique to investigate stem cell three-dimensional (3D) structures. Here, we summarized the fundamental characteristics of stem cells via 3D imaging methods and also discussed the emerging literatures on 3D imaging in stem cell research and the applications of both classical 2D imaging techniques and 3D methods on stem cells biology.

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“…The skin is a complex tissue, consisting of many layers with randomly distributed inhomogeneous light scatters such as melanin dust, melanosomes and high‐order collagen structures and elastic fibers . Although for many years in vivo optical clearing of skin using biocompatible agents such as glycerol, DMSO, and PEG400 remained in the center of interest, a few modern methods have already been applied to test fixed tissue imaging and immunolabeling performance . Liang et al .…”

Section: Application Of Toc For Particular Peripheral Organsmentioning

confidence: 99%

“…Although for many years in vivo optical clearing of skin using biocompatible agents such as glycerol, DMSO, and PEG400 remained in the center of interest, a few modern methods have already been applied to test fixed tissue imaging and immunolabeling performance . Liang et al . described CUBIC protocol suitable for whole‐mount clearing and staining allowing single cell resolution confocal investigation of, for example, basal and proliferating keratinocyte populations in the interfollicular epidermis and in the hair follicle, 300 µm deep into the tissue ( Figure e).…”

Section: Application Of Toc For Particular Peripheral Organsmentioning

confidence: 99%

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Advances in Ex Situ Tissue Optical Clearing

Matryba

Kaczmarek

Gołąb

2019

Laser & Photonics Reviews

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Examination of whole organs with subcellular resolution in health, disease, and during development is necessary to decipher their biological complexity. However, until recently, this has been virtually impossible due to the natural opacity of organ tissue. Recent progress in tissue optical clearing (TOC) has overcome this limitation by turning organs into transparent, light-permitting specimens. At least 20 original TOC methods have been developed in less than a decade, which were followed by hundreds of attempts that were aimed at their optimization and practical application. The majority of proof-of-concept studies have focused on the brain. However, it is apparent that TOC might be equally valuable when applied to peripheral organs or even the whole body. The progress in TOC for peripheral organs is delineated in an organ-by-organ fashion and whole-body clearing approaches are discussed. Additionally, physical and optical approaches for TOC affecting the optical properties of the samples and image quality are discussed to explore their advantages, limitations, and future possibilities.

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

Cited by 7 publications

References 13 publications

Neuroprotection and Axonal Regeneration Induced by Bone Marrow Mesenchymal Stromal Cells Depend on the Type of Transplant

Neuroprotection and Axonal Regeneration Induced by Bone Marrow Mesenchymal Stromal Cells Depend on the Type of Transplant

Three-Dimensional Imaging in Stem Cell-Based Researches

Advances in Ex Situ Tissue Optical Clearing

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