Preparation of Organotypic Slice Cultures from a Rat Pup's Hippocampus

Take a rat pup's brain and separate the brain hemispheres.

View the medial face of one brain hemisphere. 

Identify the fornix, a white matter band located at the medial edge of the hippocampus, and cut through it.

Separate the surrounding tissue, exposing the hippocampus and fimbria, a curved structure of white matter tracts.

Slide a spatula under the fimbria, lift the hippocampus, and transfer it to an ice-cold buffer.

Place the hippocampi from both hemispheres on a tissue chopper and slice them.

Transfer the slices to an insert membrane placed within a culture plate containing media.

Adjust the slice positions to allow nutrient and oxygen diffusion. Remove excess buffer to prevent dilution of the culture media.

Incubate. The insert membrane coating facilitates the adhesion of the slices, reducing tissue damage.

Change the media regularly to replenish nutrients, facilitating hippocampal neurogenesis and the generation of organotypic slice cultures.