Mouse anti eve

Dissections, fixations, and immunostainings were performed following standard procedures, except that for stainings to detect apoptosis in the pupal CNS, only pupae within the first 10 min of puparium formation were used in order to minimize biological variability in apoptosis levels over time. Primary antibodies used were rabbit anti-GFP (1:500 in larvae and pupae, 1:1,000 in embryos; A11122; Invitrogen), rabbit anti-DsRed (1:100; 632496; Takara Bio Inc.), rabbit anti-βgal (1:5,000; Cappel), mouse anti-Eve (1:5–1:10; 2B8; Developmental Studies Hybridoma Bank), mouse anti-Eve (1:20; 3C10; Developmental Studies Hybridoma bank), mouse anti-pERK (1:500 in retina and 1:100 in optic lobe; 9106; Cell Signaling Technology), mouse anti-Repo (1:250; 8D12; Developmental Studies Hybridoma Bank), rabbit anti-pJNK (1:200; V7931; Promega), rabbit anti-Myd88 (1:250; a gift from S. Wasserman), and rabbit anti-Dcp1 (cleaved Drosophila Dcp1 [Asp216]; 1:500; 9578S; Cell Signaling Technology). Secondary antibodies were directly conjugated Alexa Fluor 488, 546, and 647 (1:250, Molecular Probes) or biotinylated mouse or rabbit (1:300) followed by avidin amplification using the Vectastain ABC Elite kit (Vector Laboratories) or the Tyramide Signal Amplification kit (T20922; Thermo Fisher Scientific), using the manufacturer’s instructions. For sample sizes, see Table S2.

We immunostained embryos following standard protocols [32 ]. Briefly, we fixed embryos in a one-to-one mixture N-heptane and 4% formaldehyde diluted in PEM buffer (0.1 M Pipes, 1 mM MgS0₄, 2 mM EGTA) and rocked them in at room temperature for 22 min. The fixative solution was subsequently removed, and embryos were devitellinized by vigorous shaking in a 1:1 mixture of methanol:heptane. Devitellinized embryos were washed with PBS and incubated overnight at 4 °C in primary antibodies diluted in PBS-0.1% Tween 20 (PBT). After washing, embryos were incubated at room temperature in secondary antibodies for 1.5 h, and streptavidin for 20 min. The following primary antibodies were used: Mouse anti-Eve (clone 3C10, 1:50, Developmental Studies Hybridoma Bank (DSHB)), rat anti-HA (#11,867,423,001, clone 3F10, 1:500, Sigma), rabbit anti-lamin (1:2000, kind gift from Dr. Paul Fischer); rat anti-worniu (#196,362,clone 5A3AD2, 1:200, Abcam), rat anti-dpn (#195,172, clone 11D1CH11, 1:200, Abcam), mouse anti-PH3 (#14,955, 1:1000, Abcam), rabbit anti-Hb (1:200, kind gift from Dr. Chris Doe), rat anti-Zfh2 (1:200, kind gift from Dr. Chris Doe), mouse anti-AbdB (clone 1A2E9, 1:30, DSHB). Secondary antibodies against mouse, rabbit, and rat were conjugated to Alexa 488, 555, and 647 (Thermo Fisher Scientific).

For immunohistochemistry, embryos were fixed and stained according to standard procedures. Guinea pig anti-SIMU antibodies [42 (link)] was used at a 1:5000 dilution, while rabbit anti-Htl antibodies, a gift from T. Kojima, were used at a 1:1000 dilution. Rabbit anti-activated Dcp-1 (Cell signaling) and mouse anti-GFP (Roche) antibodies were used at a 1:100 dilution each. Mouse anti-REPO, mouse anti-Eve and mouse anti-CUT (Developmental Studies Hybridoma Bank) antibodies were used at 1:20, 1:100 and 1:5 dilutions, respectively. Fluorescent secondary antibodies (Cy3- and Cy5-labeled antibodies from Jackson ImmunoResearch and Alexa Fluor 488-labeled antibodies from Molecular Probes) were used at 1:200 dilutions each. A 75% glycerol solution was used as imaging medium. Microscope imaging and data analyses of all images were acquired on a Zeiss AxioImager M2 microscope equipped with an ApoTome2 optical sectioning device. All images taken using Zen acquisition software and processed with Adobe Photoshop CS6.