Drosophila Cell Transfection and Immunostaining

Drosophila S2 cells (R69007, Thermo Fisher Scientific) were cultured in Schneider’s Drosophila Media (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and penicillin-streptomycin-glutamine (Thermo Fisher Scientific) in 75 cm² flasks. The actin-Gal4 [77 (link)] and tubulin-Gal4 [78 (link)] plasmids were used as in previous publications. Cells were transfected with 1 μg of each plasmid DNA with the Effectence transfection kit (Qiagen). After 48 hours transfection, cells were fixed in 4% paraformaldehyde and mounted on coverslips coated in 0.5mg/mL Concanavalin A and ProLong Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific).
Fly adult brains and third instar wing discs were dissected, fixed, and immunostained as previously described [4 (link), 77 (link)]. The LacZ primary antibody was used at 1:500 dilution (A-11132, Thermo Fisher Scientific). Repo (8D12) and Elav (7E8A10) co-staining were performed using a 1:10 dilution (Developmental Studies Hybridoma Bank). DyLight 405, Alexa Fluor 488, and Alexa Fluor 647 conjugated secondary antibodies were used at 1:100 dilution against Repo and Elav antibodies and at 1:500 against LacZ (Jackson ImmunoResearch). All S2 cell and fly brain images were acquired on a Zeiss LSM 800 confocal microscope and processed in the Zeiss ZEN software package.