Immunofluorescence Staining of Drosophila Brains for the Single-Cell Imaging of Glial Cells

Begin with brains from transgenic Drosophila flies, containing different glial cell types harboring a cell-specific recombinase system.

It drives the expression of a cell surface protein fused to different antigenic epitopes on the same type of cells.

Treat the tissue with a fixative to cross-link proteins and preserve the tissue architecture.

Wash to remove excess fixative.

Add blocking proteins to mask non-specific binding sites to reduce background staining.

Incubate the tissue with a primary antibody cocktail that binds to the different epitopes expressed on glial cells.

Wash to remove unbound primary antibodies.

Incubate with fluorophore-conjugated secondary antibodies that bind to the primary antibodies.

Wash to remove unbound secondary antibodies.

Mount the brain inside an imaging spacer and place a coverslip. The spacer creates a chamber for the tissue, preserving its three-dimensional structure.

Different cells of the same glial type labeled with different fluorophore-conjugated antibodies allow an understanding of cell-cell interactions.

Transfer the isolated brains using a P10 pipette tip into a 200-microliter microcentrifuge tube with fixative solution. Never handle the brains using forceps. Incubate the tissues protected from light. Avoid aspirating the brains during solution transfers.

To remove the fixative, use three or more 15-minute washes with washing solution. Then, block the tissues with blocking solution for 30 minutes or longer. Next, replace the blocking solution with primary antibodies diluted in wash solution, and incubate the brains overnight at 4 degrees Celsius.

To remove the primary antibodies, use three 1-hour washes in wash solution. Next, add secondary fluorophore conjugated antibodies in wash solution, and incubate the brains overnight at 4 degrees Celsius, or for 4 hours at room temperature. To completely wash off the unbound antibodies, use three 1-hour washes in wash solution, followed by a longer wash with PBS.

Then, mount the brains. Prepare two coverslips with an imaging spacer. In the spacer, and on the extra coverslip, apply 10 microliters of mounting medium with an anti-fade agent. Next, using a P10 pipette, transfer the brains to the coverslip, depositing them next to the medium along with some PBS. Do not let the tissue dry out.

Now, carefully move the brains into the mounting medium, using the pipette. And finally, move them to the medium in the spacer and arrange them with forceps. Then, remove the adhesive liner on the spacers and attach a coverslip. Secure the coverslip with a little pressure using forceps and proceed immediately with imaging.