This article describes a method for visualizing the radial migration of granule cells in cerebellar slices from postnatal rat pups using fluorescent dyes and confocal microscopy. The protocol includes steps for preparing tissue slices, labeling cells, and capturing time-lapse images to analyze cell migration.
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 samples from light, cover the plate with foil. Then put the plate on a mutator set to 35 RPM. Let the plate incubate for 10 minutes at room temperature to let the dye label the cells.
Now, transfer the slices as before to the membrane of a transwell insert that has three micron pores. Then aspirate the loading medium, leaving just the slices. Now, remove the insert and slices to fill the well with 1.9 milliliters of DMEM. Then put the insert back and add another 100 microliters of DMEM to cover the tissues.
Incubate this preparation for two hours, after which granule cells will be visible. Transfer the plate without the lid to an environmental chamber with constant gas flow on a confocal macroscope. Then, place a glass cover on the plate insert of the confocal macroscope. For time lapse experiments, let the cells incubate in the chamber for two additional hours before proceeding.
To visualize the granule cells migrating in the tissue slices, illuminate the preparation with 488 nanometer light and use a 2X dry objective. Detect fluorescence emissions from 500 to 530 nanometers. Using ImageJ, for each image of the time lapse movie, perform a Z stack projection using the standard deviation mode.
Adjust the contrast and the brightness levels of each image so the labeled granule cells are easy to see. Now, choose the Manual Tracking plugin from the particle analysis menu. Use the plugin to click on the central point of each labeled cell body throughout the time lapse image sequence. Then export the raw tracking data to a spreadsheet for further analysis.
Reorganize the exported raw tracking data from ImageJ with a smart home made tool that identifies each cell and associated positions. Using the program, calculate the total traveled distance and the average speed of migration for each cell.
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