Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

Begin with a cerebellar slice obtained from a postnatal rat pup brain in a multi-well plate containing media.

Remove the media and incubate the slice in a cytoplasmic fluorescent dye solution to label the granule cells (GCs).

Transfer the slice onto a transwell insert membrane and remove any excess dye.

Remove the insert and fill the well with media. Then, replace the insert and add media to cover the tissue.

Incubate to allow the tissue to attach to the insert membrane.

Transfer the plate to an incubator attached to a confocal macroscope and place a glass cover over the plate.

Set the culture conditions, incubate the tissue further, then begin imaging.

Upon laser illumination, the labeled GCs fluoresce.

Capture time-lapse images to visualize the radial migration of the GCs through the molecular layer of the cerebellum.

Using imaging software, analyze the migratory distance of the GCs.

After slicing the specimen, use a truncated white bulb pipette to transfer them with some HBSS to a six-well plate. Load up to three slices in each well. Next, after emptying the media in each loaded well, add five milliliters of loading solution containing 10 micromolar of the fluorescent dye. To protect the