This article describes a detailed protocol for isolating and preparing hippocampal and rhinal cortex slices from rat pup brains. The method emphasizes maintaining structural integrity during dissection and subsequent culture.
Take a freshly harvested rat pup brain, dorsal side up, in a culture plate with chilled buffer.
Open the brain hemispheres and remove the tissues covering the hippocampus.
Incise the hemispheres so that they contain the hippocampi and rhinal cortices.
Place both hemispheres with the hippocampi facing up and parallel on a filter paper.
Transfer this assembly to a tissue chopper with the blade perpendicular to the hemispheres.
Cut the hemispheres to the required thickness to obtain slices and transfer them to a culture plate with chilled buffer.
Carefully separate the slices and dissect them to include structurally intact hippocampal and rhinal cortex regions.
Transfer the slices to membranous inserts in a multi-well plate containing media.
Remove the excess buffer from the slices and incubate them for tissue recovery.
Refresh the media. The slices can be used for further analysis.
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.
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