Goat anti ato

Eye-antennal imaginal discs were dissected in 1X phosphate buffered saline (PBS) from the wandering third-instar larvae and fixed in 4% paraformaldehyde in PBS (fixative) for 20 minutes and washed in PBST (three times)(A. Singh et al., 2002 (link); M. Tare, Puli, Moran, Kango-Singh, & Singh, 2013 ). The tissues were stained with combination(s) of antibodies following the standard protocol. Antibodies used were rat anti-Elav (1:100, Developmental Studies Hybridoma Bank, DSHB), mouse anti-Wg (1:50, DSHB), mouse anti-Sca (1:200, DSHB), goat anti-Ato (1:100, Santa Cruz Biotechnology), guinea pig anti-Homothorax (1:200, SantaCruz Biotechnology), mouse anti-β galactosidase (1:100, Promega) and rabbit anti-Eyeless (a kind gift from Uwe Walldorf and Patrick Callaerts). The discs were washed in PBST thrice for 10 minutes. Secondary antibodies used were donkey anti-rat IgG conjugated to Cy5 (1:250), donkey anti-rabbit IgG conjugated to Cy3 (1:300) or goat anti-mouse IgG conjugated to FITC (1:200) (Jackson Laboratories). The discs were mounted in Vectashield and photo-documented on a Fluoview 3000 Laser Scanning Confocal Microscope. The image analysis and preparation of the final figures was carried out using Adobe Photoshop CS6 software.