Rat anti elav 7e8a10

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.

Imaginal discs were dissected and fixed according to standard protocols. Primary antibodies used were guinea-pig anti-Hth [74 (link)], rabbit anti-PH3 (Sigma), rabbit anti β-galactosidase (Cappel), mouse anti β-galactosidase (Sigma), mouse anti-CD2 (Serotec), rabbit anti-GFP (Molecular Probes), mouse anti-Ey (Clements et al., 2008) and rabbit anti-cyclin B [75 (link)]. Mouse anti-Eya, rat anti-ELAV (7E8A10), and mouse anti-cyclin B were from Developmental Studies Hybridoma Bank (Iowa University). Fluorescently labeled secondary antibodies were from Molecular Probes. Anti-mouse-HRP (Sigma) was used for immunoperoxidase staining. Digoxigenin labelled stg RNA probe was produced from cDNA clone LD47579 (BDGP). ImageJ was used to quantify pixel intensities (http://imagej.nih.gov/ij/).

Brains were dissected in ice-cold Ringer’s solution, washed, and fixed in fresh fixative solution (4% paraformaldehyde, 50 mM NaCl, 0.1 M Pipes, pH 7.2) for 20 min at room temperature with gentle agitation. Fixed tissues were analyzed by immunostaining and microscopy. Immunostaining was performed using fluorescent secondary antibodies essentially as described earlier (Lyalin et al., 2006 (link)). The following primary antibodies and corresponding dilutions were used for immunostaining: mouse anti-GFP 8H11 (1:100), anti-Repo 8D12 (1:10), rat anti-Elav 7E8A10 (1:10), mouse anti-Brp nc82 (1:10), all from Developmental Studies Hybridoma Bank; rabbit anti-GFP from Invitrogen (1:800), rat anti-HA from Roche (1:1000), rat anti-Dpn from Abcam (1:500). The following secondary antibodies were used: goat anti-rabbit and anti-mouse Alexa Fluor 546 and 488 (1:250), all from Invitrogen; donkey anti-mouse and anti-rabbit Cy3 (1:250) and FITC (1:150), from Jackson Laboratories. Stained samples were mounted on slides in Vectashield (Vector Laboratories) and imaged using Zeiss Axio Imager M2 fluorescence microscope with ApoTome module for optical sectioning or Zeiss 510 META Confocal microscope. Images were processed using Zeiss Zen and ImageJ software.