Visualizing Astrocyte Morphology Using Fluorescent Dye Iontophoresis in a Fixed Mouse Brain Slice

Place a chemically fixed mouse brain slice in a glass bottom dish containing buffer and secure it with a mesh.

Connect an electrode filled with an anionic, hydrophilic fluorescent dye to a voltage source.  

Insert a ground electrode into the buffer to complete the electrical circuit.

Using brightfield illumination, advance the dye-filled electrode toward the slice.

Switch to infrared differential interference contrast imaging to locate an astrocyte.

Pierce the astrocyte soma with the electrode to access the cytoplasm.

Apply an electric current to drive the dye into the cytoplasm via iontophoresis.

Switch to confocal microscopy to monitor dye movement.

The hydrophilic dye, unable to cross the hydrophobic membrane, diffuses within the cytoplasm to fill the soma and branches.

Once the cell is filled, stop the current and withdraw the electrode.

Allow the astrocyte membrane to reseal.

Acquire images at various Z-planes and reconstruct them to visualize astrocyte morphology.