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.

Transfer three to five dorsal hippocampal sections for each mouse into the wells of a 24-multi-well plate containing 500 microliters of 0.1 molar PBS for each well. Wash the sections contained in each well with 500 microliters of 0.1 molar PBS. Perform all washes and incubations at room temperature with gentle agitation, unless otherwise mentioned. Under a fume hood, incubate the sections in 40% methanol, 1% hydrogen peroxide in 0.1 molar PBS for 20 minutes in order to quench the endogenous peroxidase activity.

Following three more washes in 0.1 molar PBS, permeabilize the sections by incubating them in 500 microliters of 0.2% Triton X-100 for five minutes. While the sections are incubating, prepare enough blocking buffer for the entire experiment. Then, remove the permeabilization solution and add 100 microliters of the blocking buffer to each well. Incubate the sections for one hour.

Next, remove the blocking buffer and cover the sections with phosphorylated extracellular regulated kinase primary antibody, diluted 1 to 500 in blocking buffer. Incubate the sections overnight at 4 degrees Celsius with gentle agitation. The next day, after washing the sections three times in 0.1 molar PBS for 5 minutes each, incubate the sections with a biotin conjugated secondary antibody diluted 1 to 250 blocking buffer for one hour at room temperature.

During the incubation, prepare the ABC reagent at least 30 minutes before use in order to allow enough time for the avidin and biotin complex to form. After washing the sections three more times with 0.1 molar PBS, add the ABC reagent, and allow it to incubate with the sample for 45 minutes. During the next set of three washes in PBS, prepare the dab working solution in the fume hood according to the manufacturer's specifications. Aliquot the dab working solution in the plate.

Then, transfer sections into the wells containing the DAB working solution, and slowly swirl the plate to allow maximum exposure of the sections to the solution. Monitor the DAB reaction on a microscope until adequate signal develops in about two to five minutes. Stop the reaction at the desired color intensity by washing the tissue in 0.1 molar PBS. After three more washes in PBS, mount the sections on gelatin coated slides, and dry them at room temperature for at least three to four hours.

Once air dried, transfer slides in the fume hood, and dehydrate the sections by sequentially placing them into graded ethanol solutions for two minutes each. Then, clear the slides in 100% xylenes for five minutes. Coverslip the slides using mounting medium, and leave them in the fume hood overnight to dry.

Transfer the slides to a bright field microscope equipped with a camera in 20x objective lens. Acquire multiple brightfield images from each section at 200x magnification, covering the dorsal hippocampus. Image three to five sections from each animal.