Imaging Hippocampal Neuronal Activation In Mouse Brain Sections

Start with mouse brain sections exposing the dorsal hippocampus, rich in neurons.

Add hydrogen peroxide to quench neuronal peroxidase activity, followed by washing. 

Treat with a permeabilization buffer, then apply a blocking buffer to block non-specific binding.

Add the primary antibodies and incubate with agitation. These antibodies bind to neuronal activation marker proteins.

Wash and apply biotin-conjugated secondary antibodies to bind the primary antibodies. 

Rinse and introduce an avidin-biotin-peroxidase complex that binds to biotin on the conjugates. 

Wash again, then add a chromogenic substrate, which is oxidized by peroxidase to generate a brown precipitate. Stop the reaction with PBS.  

Transfer the sections to a gelatin-coated slide and dehydrate them using increasing concentrations of ethanol. Then, clear the sections in xylene and mount them.

Place the slide under a bright-field microscope.

Under light illumination, unstained regions transmit light and appear bright, while stained neurons absorb and reflect light and appear darker. 

The distinct darker area indicates neuronal activation within the hippocampus.