Preparing Rhinal Cortex-Hippocampus Slices from a Rat Pup Brain

Transfer the brain, dorsal side up, into a 60-millimeter plate containing 5 milliliters of cold GBSS. Insert fine forceps into the cerebellum, and insert the spatula along the midline to carefully open the hemisphere. Use short, curved forceps to carefully remove the excess tissue that covers the hippocampus without touching the hippocampal structure. Then, cut below each hippocampus.

Transfer one hemisphere at a time, hippocampus side up and parallel to each other, onto a piece of filter paper. Place the filter paper onto a tissue chopper with the hemispheres perpendicular to the blade, and cut the hemispheres into 350-micron slices. Transfer the sliced tissue into a new Petri dish containing 5 milliliters of cold GBSS, and use round-tip electrodes to carefully separate the slices.

Keeping only the samples with a structurally intact rhinal cortex and hippocampus, use a spatula and a round-tip electrode to place up to four intact slices onto individual inserts in the appropriate number of wells of a six-well plate containing 1.1 milliliters of culture medium per well.

Use a P20 pipette to remove any excess dissection medium from around each slice. Place the plate in the cell culture incubator. Change the medium every two to three days. Use forceps to lift the insert. Aspirate the medium from the corresponding well, and place the insert back in place. Use a P1000 pipette to add 1 milliliter of fresh 37 degrees Celsius medium to each well.