Analyzing Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures
Transcript
Begin with a mouse pup brain.
Isolate the cerebellum and obtain tissue slices of appropriate thickness.
Transfer the slices onto membrane inserts placed within culture plate wells containing media.
In treated wells, the media contains a neuroprotective test compound. Control wells lack the compound. Incubate.
Under control conditions, the cerebellar Purkinje cells undergo apoptosis, mimicking early postnatal development in vivo.
In treated wells, depending on its potential, the compound may protect the cells from apoptosis.
Wash the slices and fix them to preserve cellular integrity.
Transfer the slices into a permeabilization-blocking solution to permeabilize cell membranes and block non-specific binding sites.
Overlay with primary antibodies targeting a calcium-binding protein specifically expressed in Purkinje cells.
Wash and introduce fluorophore-conjugated secondary antibodies targeting the primary antibodies.
Counterstain the nuclei with a DNA-binding dye and mount the slices.
Using a confocal microscope, quantify the viable Purkinje cells to identify the test compound's neuroprotective potential
