Neuronal Death Assay to Evaluate Cell Death in Ex Vivo Epilepsy Model
Transcrição
To perform the propidium iodide uptake assay, work in a biosafety cabinet.
Lift the insert from the well, and add propidium iodide in medium to a final concentration of 2 micromolar per well. Slowly agitate the plate before placing the insert back into the well, taking care that there are no bubbles beneath the slices. When all slices have been treated, return the plate to the cell culture incubator.
For immunostaining of the slices, at the end of the propidium iodide incubation, aspirate the medium from the bottom of each well, and add 1 milliliter of 4% paraformaldehyde to the top and bottom of each insert.
After one hour at room temperature, wash the slices 2 times for 10 minutes and 1 milliliter of PBS per wash, and use a hydrophobic pen to draw two rectangles on microscope slides.
Use a sharp blade to cut the slices from the inserts and place one slice into each rectangle. Add permeabilization/blocking solution onto each slice for a three-hour incubation at room temperature. At the end of the incubation, add the primary antibodies of interest to each slice for a 4-degree Celsius incubation overnight.
The next morning, wash the slices with PBS-Tween three times for 10 minutes per wash, and incubate the slices with the appropriate secondary antibodies for four hours at room temperature. Afterwards, wash the slices as demonstrated, and add 50 microliters of Hoechst solution to each slice for a 20-minute incubation at room temperature.
Following Hoechst incubation, wash the slices again, and add 50 microliters of mounting medium to each one. Then, place a coverslip over the slices and seal with nail polish.
After allowing the slides to dry for 24 hours at room temperature, visualize the immunostaining and propidium iodide uptake by confocal microscopy.
