Investigation of PRDM10 and PRDM13 Expression in Developing Mouse Embryos by an Optimized PACT-Based Embryo Clearing Method

Recent developments in tissue clearing methods have significantly advanced the three-dimensional analysis of biological structures in whole, intact tissue, providing a greater understanding of spatial relationships and biological circuits. Nonetheless, studies have reported issues with maintaining structural integrity and preventing tissue disintegration, limiting the wide application of these techniques to fragile tissues such as developing embryos. Here, we present an optimized passive tissue clearing technique (PACT)-based embryo clearing method, initial embedding PACT (IMPACT)-Basic, that improves tissue rigidity without compromising optical transparency. We also present IMPACT-Advance, which is specifically optimized for thin slices of mouse embryos past E13.5. We demonstrate proof-of-concept by investigating the expression of two relatively understudied PR domain (PRDM) proteins, PRDM10 and PRDM13, in intact cleared mouse embryos at various stages of development. We observed strong PRDM10 and PRDM13 expression in the developing nervous system and skeletal cartilage, suggesting a functional role for these proteins in these tissues throughout embryogenesis.

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EZ Clear for simple, rapid, and robust mouse whole organ clearing

10.1101/2022.01.12.476113 ◽

2022 ◽

Author(s):

Chih-Wei Hsu ◽

Juan Cerda ◽

Jason M Kirk ◽

Williamson D. Turner ◽

Tara L. Rasmussen ◽

...

Keyword(s):

Biomedical Research ◽

Room Temperature ◽

Adult Mouse ◽

Dimensional Space ◽

Three Dimensional ◽

Fluorescence Signal ◽

Tissue Architecture ◽

Tissue Clearing ◽

Clearing Method ◽

Three Dimensional Space

Tissue clearing for whole organ cell profiling has revolutionized biology and imaging for exploration of organs in three-dimensional space without compromising tissue architecture. But complicated, laborious procedures, or expensive equipment, as well as the use of hazardous, organic solvents prevents the widespread adoption of these methods. Here we report a simple and rapid tissue clearing method, EZ Clear, that can clear whole adult mouse organs in 48 hours in just three simple steps. Samples stay at room temperature and remain hydrated throughout the clearing process, preserving endogenous and synthetic fluorescence, without altering sample size. After wholemount clearing and imaging, EZ Cleared samples can be further processed for downstream embedding and cryosectioning followed by standard histology or immunostaining, without loss of endogenous or synthetic fluorescence signal. Overall, the simplicity, speed, and flexibility of EZ Clear make it easy to adopt and apply to diverse approaches in biomedical research.

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Three-Dimensional Imaging of the Intracellular Fate of Plasmid DNA and Transgene Expression: ZsGreen1 and Tissue Clearing Method CUBIC Are an Optimal Combination for Multicolor Deep Imaging in Murine Tissues

PLoS ONE ◽

10.1371/journal.pone.0148233 ◽

2016 ◽

Vol 11 (1) ◽

pp. e0148233 ◽

Cited By ~ 22

Author(s):

Shintaro Fumoto ◽

Koyo Nishimura ◽

Koyo Nishida ◽

Shigeru Kawakami

Keyword(s):

Plasmid Dna ◽

Transgene Expression ◽

Three Dimensional ◽

Optimal Combination ◽

Three Dimensional Imaging ◽

Tissue Clearing ◽

Deep Imaging ◽

Clearing Method ◽

Intracellular Fate

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A pH-Adjustable Tissue Clearing Solution That Preserves Lipid Ultrastructures: Suitable Tissue Clearing Method for DDS Evaluation

Pharmaceutics ◽

10.3390/pharmaceutics12111070 ◽

2020 ◽

Vol 12 (11) ◽

pp. 1070

Author(s):

Shintaro Fumoto ◽

Eriko Kinoshita ◽

Keisuke Ohta ◽

Kei-ichiro Nakamura ◽

Tasuku Hirayama ◽

...

Keyword(s):

Spatial Distribution ◽

Three Dimensional ◽

Fluorescent Intensity ◽

Tissue Clearing ◽

Carbocyanine Dyes ◽

Electron Microscopy Level ◽

Wide Ph Range ◽

Ph Range ◽

Clearing Method ◽

And Oxidative Stress

Visualizing biological events and states to resolve biological questions is challenging. Tissue clearing permits three-dimensional multicolor imaging. Here, we describe a pH-adjustable tissue clearing solution, Seebest (SEE Biological Events and States in Tissues), which preserves lipid ultrastructures at an electron microscopy level. Adoption of polyethylenimine was required for a wide pH range adjustment of the tissue clearing solution. The combination of polyethylenimine and urea had a good tissue clearing ability for multiple tissues within several hours. Blood vessels stained with lipophilic carbocyanine dyes were deeply visible using the solution. Adjusting the pH of the solution was important to maximize the fluorescent intensity and suppress dye leakage during tissue clearing. The spatial distribution of doxorubicin and oxidative stress were observable using the solution. Moreover, spatial distribution of liposomes in the liver was visualized. Hence, the Seebest solution provides pH-adjustable, rapid, sufficient tissue clearing, while preserving lipid ultrastructures, which is suitable for drug delivery system evaluations.

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