Immunostaining of Drosophila Eye and Brain

Embryos and larval eye discs, wing discs and brains were stained as described [93 (link), 94 (link)], fixing 30 min in 4% formaldehyde in 0.1M PIPES pH 7.0/2mM MgSO₄/1mM EGTA for most antibodies but 45 min in 2% formaldehyde in 75mM lysine/370mM sodium phosphate pH 7.2/10mM NaIO₄ for anti-Gl. Pupal retinas were fixed and stained as described [95 (link)]. Antibodies used were mouse anti-Gl (1:10; Developmental Studies Hybridoma Bank (DSHB), mouse anti-Chp (1:50; DSHB), chicken anti-GFP (1:300; Aves), rat anti-Elav (1:100; DSHB), mouse anti-Cut (1:10; DSHB), rabbit anti-β-galactosidase (1:5000; Cappel), rat anti-Ecad (1:10; DSHB), mouse anti-FasIII (1:10; DSHB), rat anti-Ncad (1:10; DSHB), rat anti-Kettin/Sls (1:200; Abcam), mouse anti-Eya (1:10; DSHB), mouse anti-Lz (1:10; DSHB), mouse anti-Pros (1:10; DSHB), rabbit anti-Hth (1:200) [96 (link)], rat anti-Pax2 (1:500) [24 (link)] and mouse anti-Arm (1:10; DSHB). Secondary antibodies were from Jackson Immunoresearch (Cy3 or Cy5 conjugates used at 1:200) or Invitrogen (Alexa488 conjugates used at 1:1000). Images were captured on a Leica SP5 confocal microscope and processed using ImageJ and Adobe Photoshop. Cone cell counts were performed using ImageJ.