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