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.