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.

For the dye injection test, gently place a lightly fixed brain slice into a glass bottom dish filled with 0.1 molar PBS at room temperature. And hold the slice in place with a platinum harp with nylon strings.

Connect the electrode to a voltage source and place the ground electrode into the bath containing the brain slice. Move the objective of a confocal microscope to the brain region of interest.

And lower the electrode into the solution using the 10x water immersion lens, moving the electrode to the center of the field of view. Then, under bright field, slowly lower the electrode toward the slice, stopping just above the surface.

To fill an astrocyte of interest with iontophoresis, use the infrared differential interference contrast to identify astrocytes with elongated, oval-shaped somata, about 10 micrometers in diameter, 40 to 50 micrometers below the slice surface.

This is the stratum radiatum of the hippocampus directly below the pyramidal cell layer. As we move through the tissue, we can see blood vessels, neurons, astrocytes, and other cell types.

Once an astrocyte has been selected, move the cell to the center of the field of view and slowly lower the electrode tip into the slice. Navigating through the tissue until the electrode is on the same plane as the cell body.

Once the cell body of the astrocyte is clearly visible and outlined, slowly and gently advance the electrode forward, moving the electrode until the tip impales the soma of the cell.

Move the focus of the objective slowly up and down to note if the electrode is inside the soma. Once the electrode tip is inside the cell, turn on the stimulator at 0.5 to 1 volt to continuously eject current into the cell.

Using the confocal microscope, watch the cell fill, increasing the digital zoom to visualize the details of the cell, and to make sure that the tip of the electrode is visible inside the cell as necessary.

Wait for about 15 minutes until the finer branches and processes appear defined, before turning off the voltage and gently withdrawing the electrode tip from the cell.

To image the filled cell, wait 15 to 20 minutes for the cell to return to its original form, before adjusting the setting on the confocal microscope to make sure that the finer branches and processes appear defined under the 40x objective.

Set up a z-stack with a step size of 0.3 micrometers, moving the objective while imaging until there is no signal from the cell. And set that step as the top.

Then move the objective down, focusing through the cell until there is no signal. And set that step as the bottom.