Cy3 conjugated donkey anti mouse igg

Staining of larval tissues was performed as described previously^{38 (link),44 (link)}. Larvae were dissected in PBS, fixed with 4% formaldehyde for 40 minutes on ice and then permeabilized for 15 minutes at room temperature in PBS containing 0.5% NP-40. The following primary antibodies were used in overnight incubations at 4 °C in blocking solution: rabbit anti-Apt (1:1000), rabbit anti-β-galactosidase (1:2000, Cappel), rabbit Caspase3 (1:50, Cell Signaling Technology), mouse anti-β-galactosidase (1:500, Sigma), mouse anti-Ubx (1:10, Developmental Studies Hybridoma Bank (DSHB)), goat anti-Cyclin E (1:200, Santa Cruz). The secondary antibodies used were as follows: Alexa 488 donkey anti-rabbit IgG conjugate (1:500, Molecular Probes), Alexa 488 donkey anti-mouse IgG (1:500, Molecular Probes), Cy3-conjugated donkey anti-mouse IgG (1:500, Sigma), Cy3-conjugated goat anti-rabbit IgG (1:500, CWBIO), bovine anti-goat IgG-CFL 555 (1:500, Santa Cruz). Mounting used VECTASHIELD Mounting Medium with DAPI (Vector Labs). The caspase-3 staining was did as described previously^{45 (link)}.

Oocytes isolated from antral follicles were cleared of the zona pellucida in acid Tyrode’s solution 1 h–1 h 30 before fixation. Immunostaining was performed using a modified protocol from Reference 33 (link). Oocytes were fixed and permeabilized 6 h after NEBD in 1X PBS, Hepes (100 mM, pH 7), EGTA (50 mM, pH 7), and MgSO₄ (10 mM) buffer with 0.2% Triton X-100 and 2% formaldehyde for 30 min at 37°C. Oocytes were then washed in 1X PBS and 0.1% Triton X-100 (PBSTr thereafter) where they were stored overnight at 4°C. The remaining immunostaining protocol is similar to that for phalloidin TZP labeling for spinning disk and OMX microscopy, except that oocytes were directly blocked the day after fixation and blocking and antibody incubations were performed in PBSTr and 3% BSA. Oocytes were mounted on slide wells filled with VECTASHIELD Antifade Mounting Medium with DAPI (Ref. H-1200; Vector Laboratories). We used mouse anti-α-tubulin (1:200, Ref. T8203; Sigma-Aldrich) as primary antibodies and Cy3-conjugated donkey anti-mouse IgG (1:150, Ref. 715-165-151; Jackson ImmunoResearch) as secondary antibodies. Incubation with Alexa Fluor 488–conjugated phalloidin (10 U/ml, Ref. A12379; Thermo Fisher Scientific) was performed along with secondary antibody incubation.

The antibodies were used at the following dilutions: rabbit anti-Mid (1:50050 (link)), mouse anti-Wg (1:50 DSHB), mouse anti-Arm (1:200 DSHB), rabbit anti-Hh (1:800 gift of T. Tabata), mouse anti-40-1a (1:500 DSHB), 488 donkey anti-rabbit IgG conjugate (1:500, Alexa), Cy3–conjugated donkey anti-mouse IgG (1:500, Sigma), TOTO-3(1:200, Probes) and cleaved Caspase-3(1:200, Cell Signaling).
Images were acquired under Leica TCS SP5 confocal microscope, Olympus DP2-BSW microscope and Olympus cellSens, processed using Adobe Photoshop CS6.