JoVE Encyclopedia of Experiments
Neuroscience
0 views • 5:06 min • July 8th, 2025
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 le
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