Neuronal Death Assay to Evaluate Cell Death in Ex Vivo Epilepsy Model
Transcript
Epilepsy — spontaneous and recurrent seizures in the brain — leads to neuronal cell death.
To study neuronal death in an ex vivo epilepsy model, begin by taking a rhinal cortex-hippocampus organotypic slice — prepared from a rat pup brain — in an appropriate medium.
The organotypic slice consists of the rhinal cortex and hippocampus. Under gradual serum deprivation, these regions depict evolving epileptic events, leading to seizure-induced neuronal death.
Add the required amount of propidium iodide, PI — a fluorescent dye — to the culture and incubate. Through the damaged cell membrane, PI enters the dead neurons and binds to DNA; the live neurons, with intact membrane, are not capable of PI uptake.
Place the organotypic slice on a microscope slide for immunostaining. Add permeabilization-blocking solution and incubate.
Detergent molecules in the solution solubilize membrane lipids, creating pores in the membrane of intact cells — rendering them permeable for subsequent immunostaining steps. Proteins in blocking solution passively bind to non-specific sites of the cells, reducing background staining.
Add a primary antibody solution that penetrates inside the neurons and binds to the neuron-specific nuclear antigen — NeuN — distributed in the nuclear matrix. Add fluorescently-labeled secondary antibodies that bind to the primary antibodies.
Place mounting medium on the slide to stabilize the specimen and seal with a coverslip. Under a confocal microscope, cells showing dual staining — NeuN-stained neurons which are PI-positive — indicate neuronal death.
