All procedures (TEM and Immunofluorescence) were performed as previously described (Nie et al. 2012 (link), 2014 (link)). For TEM, heads taken from flies 1–2 days of posteclosion. The antibodies used in this study were: rat anti-Elav (7E8A10, 1:100, Developmental Studies Hybridoma Bank) primary antibody, mouse anti-EYS (21A6, 1:50, Developmental Studies Hybridoma Bank), rabbit anti-Rh6 (1:1,000, Dr. Claude Desplan), mouse anti-Rh5 (1:50, Dr. Steve Britt), Cy5 conjugated donkey antirat secondary (1:200, Jackson Immunoresearch #712-175-153), Alexa 488 conjugated goat antirabbit (1:200, Life Technologies #A-11008), Alexa 488 conjugated goat antimouse (1:200, Jackson Immunoresearch #115-545-146), and Cy5 conjugated goat antimouse (1:200, Jackson Immunoresearch #115-175-166). F-actin was labeled using rhodamine-conjugated phalloidin (1:200, Life Technologies #R415). Confocal images were captured on a Leica TCS SP5. TEM imaging was conducted with a JOEL 1010 and JOEL 1400. All images were processed in Fiji or Adobe Photoshop.
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