Immunostaining of Drosophila Imaginal Discs

Eye-antennal imaginal discs were dissected from the third instar larvae in cold 1× phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde in 1× PBS for 20 min, and then quickly washed once in 1× PBS. It was followed by three washes in 1× PBST. The tissues were stained with a combination of antibodies following a previously published protocol [42 (link), 52 (link)]. The primary antibodies used were rat anti-Embryonic Lethal Abnormal Vision (ELAV) (1:100; Developmental Studies Hybridoma Bank, DSHB), mouse anti-Discs-large (Dlg) (1:100; DSHB), and mouse anti-Chaoptin (24B10) (1:100; DSHB) [53 (link)]. Secondary antibodies (Jackson Laboratory) used were goat anti-rat IgG conjugated with Cy5 (1:250), and donkey anti-mouse IgG conjugated with Cy3 (1:250). The tissues were mounted in the antifading agent: Vectashield (Vector Laboratories). The immunofluorescent images were captured at 20× magnification by using Olympus Fluoview 3000 Laser Scanning Confocal Microscope [54 ]. All final figures were prepared using Adobe Photoshop software.