Mouse anti eya

Immunohistochemistry was performed as previously described [34 (link)], except in the case of dpERK antibody, for which testes were dissected and fixed in 10 mM Tris-HCl, pH 6.8, 180 mM KCl, 50 mM NaF, 10 mM NaVO₄ and 10 mM β-glycerophosphate as described in [26 (link)] and then treated as in the case of other antibodies. We used the following primary antibodies: guinea pig anti-Tj (1:3000, gift of D. Godt), rabbit anti-Zfh1 (1:5000, gift of R. Lehmann), rabbit anti-Stat92E (1:1000), rabbit anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (1:200, Cell Signaling #9101), mouse anti-βPS Integrin, mouse anti-Eya, mouse anti-Fas3 (all 1:20, Developmental Studies Hybridoma Bank (DSHB), created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242), goat anti-Vasa (1:100, Santa Cruz Biotechnology), rabbit anti-GFP (1:500, Life Technologies), chicken anti-GFP (1:500, Aves Labs).

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/).