Immunostaining, dehydration, and clearing of mouse embryos for ultramicroscopy
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Abstract:
This protocol describes the preparation of mouse embryos for ultramicroscopy (UM), a powerful imaging technique that achieves precise and accurate three-dimensional (3D) reconstructions of intact macroscopic specimens with micrometer resolution. In UM, a specimen in the size range of ∼1-15 mm is illuminated perpendicular to the observation pathway by two thin counterpropagating sheets of laser light. In combination with fluorescein isothiocyanate (FITC) immunostaining, UM allows visualization of somatic motor and sensorial nerve fibers in whole mouse embryos. Even the fine branches of the sensomotoric fibers can be visualized over a distance of up to several millimeters. In this protocol, mouse embryos are fixed and immunostained in preparation for UM. Because UM requires the excitation light sheet to travel throughout the entire horizontal width of the specimen, specimens usually have to be rendered transparent before microscope inspection. Here, the embryos are dehydrated in ethanol and then cleared in a solution of benzyl alcohol and benzyl benzoate.
Insights
This protocol details preparing mouse embryos for ultramicroscopy (UM), enabling 3D visualization of nerve fibers. The method uses specific clearing techniques for enhanced imaging of fine neural structures.
Area of Science:
- Developmental biology
- Neuroscience
- Microscopy techniques
Background:
- Ultramicroscopy (UM) offers high-resolution 3D imaging of macroscopic specimens.
- Visualizing fine neural structures in whole embryos presents significant challenges.
- Existing methods may lack the resolution or penetration for detailed embryonic nerve imaging.
Purpose of the Study:
- To establish a protocol for preparing mouse embryos for ultramicroscopy.
- To enable detailed visualization of somatic motor and sensorial nerve fibers in whole mouse embryos.
- To optimize sample clearing for effective light sheet penetration in UM.
Main Methods:
- Mouse embryos were fixed and processed using fluorescein isothiocyanate (FITC) immunostaining.
- Specimens underwent dehydration in ethanol.
- Clearing was performed using a benzyl alcohol and benzyl benzoate solution to render embryos transparent.
Main Results:
- The protocol successfully prepared mouse embryos for ultramicroscopy.
- Somatic motor and sensorial nerve fibers, including fine branches, were visualized in 3D.
- UM achieved micrometer resolution, allowing visualization of neural structures up to several millimeters.
Conclusions:
- This protocol provides a reliable method for preparing mouse embryos for ultramicroscopy.
- The optimized clearing technique facilitates deep tissue imaging of neural networks.
- UM is a powerful tool for studying embryonic neuroanatomy and development.
