Labeling of Biocytin-Filled Interneurons in Rat Hippocampal Slices for Visualization

Take fixed and permeabilized rat hippocampal slices containing biocytin-filled interneurons with inactivated endogenous peroxidases.

Add avidin-biotin complexes (ABC) containing horseradish peroxidase (HRP) and incubate, allowing the avidin to bind to biocytin.

Wash off unbound ABC.

Add a mixture of chromogenic substrate and metal enhancer, then incubate.

Introduce hydrogen peroxide to activate the HRP, which oxidizes the substrate to form a dark precipitate, while the enhancer improves contrast.

Transfer the slices to filter paper and remove excess buffer. Treat with osmium tetroxide to enhance the contrast.

Place the slices on a slide and mount. Dehydrate them using increasing ethanol concentrations.

Wash the slices in absolute ethanol and propylene oxide.

Add resin to infiltrate the tissues.

Place the slices into a planchette and heat to polymerize the resin. Transfer the slices to a slide, place a coverslip, and solidify the resin.

Using light microscopy, visualize the darkly stained biocytin-filled interneurons.

Place the sections into a clean glass vial containing PBS. Perform the avidin HRP reaction by first incubating the sections in avidin biotin complex, or ABC, for at least two hours to amplify the HRP reaction product.

Next, wash the sections with PBS 3 times for 10 minutes, and then with Tris buffer twice for 10 minutes. After removing the last trace buffer wash, quickly add 1 drop of 8% nickel chloride solution to the diaminobenzidine solution, or DAB solution. Then pipette the solution in and out to mix.

And quickly add 1 milliliter of this solution over the sections. Incubate the sections in this solution for 15 minutes. Then, add 10 microliters of 1% hydrogen peroxide to the DAB solution. Allow the reaction to proceed in the dark under constant agitation for about 1 to 2 minutes, until the cells are labeled.

In a fume hood, place a small circle of filter paper into a Petri dish and dampen it with PB. Lift the sections one at a time from the glass vial using a paintbrush, and carefully place them flat on the paper. Cover the sections with another moistened circle of filter paper. And remove excess buffer by gently touching tissue paper to the surface.

Apply 8 or 9 drops of 1% osmium tetroxide in PB to the top paper. Cover the dish and retain in the fume hood for at least 30 minutes, but no more than one hour. After rinsing, mounting, and cover-slipping the sections, dehydrate through a series of 50%, 70%, 95%, and 100% alcohol solutions.

Following the dehydration step, transfer the sections to a glass vial containing 100% ethanol on a shaker for approximately 5 minutes. Replace the alcohol solution with propylene oxide, and wash three times for 5 minutes. Following the last wash, keep around 2 milliliters of propylene oxide in the vial, and add resin at a one-to-one ratio.

Ensure that the resin is dissolved, and keep the sections under constant agitation for 30 minutes. After 30 minutes, use a wooden stick to place each section in an aluminum planchet containing epoxy resin and incubate overnight. The next day, place the planchet on a hot plate for approximately 10 minutes.

Then, pick up each section with a wooden stick and place on a clean slide. Once all the sections have been transferred onto the slide, view the slide under a dissecting microscope to check the orientation of the slices and place a coverslip over the sections. Cure in the oven for 48 hours at 56 degrees Celsius.