Fluorescence Immunostaining of Rat Hippocampal Neurons within a Microfluidic Chip

Take a microfluidic chip containing rat hippocampal neurons.

The chip features somatic and axonal reservoirs, with compartments divided by microgrooves, effectively compartmentalizing the neurons and isolating the somatic and axonal regions.

Remove the media and add formaldehyde. Incubate to fix the neurons and preserve their structure.

Remove excess formaldehyde and wash with buffer. Then, add a detergent to permeabilize the neurons.

Replace the detergent with a blocking solution to block non-specific binding sites.

Remove the blocking solution and incubate with primary antibodies that bind to specific neuronal synaptic markers, which are integral membrane proteins on synaptic vesicles.

Remove the unbound antibodies and wash with buffer.

Incubate with fluorophore-tagged secondary antibodies that bind to the primary antibodies targeting synaptic markers.

Remove unbound secondary antibodies and wash with buffer.

Under a confocal microscope, image the chip to visualize the fluorescent neuronal synaptic markers.

To begin fluorescence immunostaining, remove most of the media from the chip, keeping the interior compartments hydrated. Immediately, add 100 microliters of fixation solution to the top wells of the axonal and somatic compartments.

After one minute, add 100 microliters of fixation solution to the bottom wells, and fix the cells for 30 minutes at room temperature. Remove most of the solution from the wells, and immediately add 150 microliters of PBS to each of the top wells of the axonal and somatic compartments. Wait two minutes for the PBS to flow into the bottom wells, then, remove the PBS and rinse the wells twice more using this same process.

Following the last rinse, remove most of the PBS from the wells of the chip, and immediately add 150 microliters of PBS with 0.25% Triton X-100 to each of the top wells of the axonal and somatic compartments.

Wait for 15 minutes, and then, remove most of the liquid from the wells. Immediately add 150 microliters of blocking solution to each of the top wells of the axonal and somatic compartments. After 15 minutes, remove most of the liquid from the wells, and immediately add 100 microliters of the primary antibody solution to each of the top wells of the axonal and somatic compartments.

Cover the chip to minimize evaporation, and incubate the cells in the primary antibody for one hour at room temperature. Next, rinse the chip by removing most of the solution, and immediately adding 150 microliters of PBS to each of the top wells of the axonal and somatic compartments. After five minutes, remove the PBS from both wells, and repeat the rinse two more times.

Now, remove most of the liquid from the wells, and immediately add 100 microliters of secondary antibody in PBS to each of the top wells of the axonal and somatic compartments. Cover the chip to minimize evaporation, and incubate the chip for one hour at room temperature. As previously shown, rinse the chip three times with PBS. Then, mount and image the chips as described in the accompanying text protocol.