Confocal Microscopy Imaging of Intact Dendritic Arbors and Spine Morphology in a Cleared Mouse Brain

Take a cleared mouse brain with cells expressing fluorescent proteins and lipids removed for transparency for deep-structure imaging with high resolution.

A hydrogel mesh preserves biomolecules and maintains neuronal networks.

Transfer the brain into an imaging chamber containing an agarose ring for support.

Secure the tissue with adhesive and fill the chamber with a refractive index matching solution to minimize light scattering and create a stable imaging environment.

Place the chamber on a confocal microscope stage and gently lower the objective into the solution to establish a continuous column of contact.

Use epifluorescence to locate regions of interest. Then, adjust the laser and imaging parameters to optimize image quality.

Capture a series of z-stacks to create a three-dimensional representation of the tissue.

Use appropriate software to analyze cellular morphology, including dendritic arbors, their spines, and the distribution and density of neurons.

To construct a large tissue imaging chamber for tissues with more than a 5 millimeter thickness, use a 10 centimeter glass dish with a high wall. Place a 50 milliliter conical tube in the center, making sure that the diameter o