Rabbit anti pjnk

For antibody staining, the lymph glands and circulating hemocytes of third-instar larvae were fixed and stained as described previously [11 (link),34 (link)]. Briefly, 5–6 third-instar larvae (96 h after egg laying) were opened via an incision at both the posterior and anterior ends in 20 μL of phosphate-buffered saline (PBS), and the circulating hemocytes were bled and allowed to attach to a glass slide for 30 min. The following primary antibodies were used: rabbit anti-GFP (1:100, Thermo Fisher Scientific, Waltham, MA, USA), mouse anti-L1 (1:50), and mouse anti-P1 (1:50), which were gifts from I. Ando; rabbit anti-p-Erk (1:50, Cell Signaling Technology, Danvers, MA, USA); and rabbit anti-p-JNK (1:200, Promega, Madison, WI, USA). Mouse anti-dorsal (1:50, Developmental Studies Hybridoma Bank, Iowa City, IA, USA); rat anti-Stat92E (made in our lab); rabbit anti-PPO1 (1:100, a gift from Erjun Ling); rabbit anti-PH3 (1:200, Millipore, Burlington, MA, USA); Alexa Fluor 488-, Alexa Fluor 568-, and Alexa Fluor 594-conjugated secondary antibodies (Thermo Fisher Scientific, USA) and Hoechst (1:500, Sigma-Aldrich, Burlington, MA, USA) were used. All staining was performed in at least three independent experiments.

Wing imaginal discs were stained using normal procedures and mounted in Vectashield (Vector Laboratories, Burlingame, CA, USA). We used the following primary antibodies: goat anti-GFP^FICT (Abcam, 1:500), rabbit anti-aPKC (Santa Cruz Biotechnology, 1:300), mouse anti-RFP (Proteintech, 1:500), rabbit anti-PH3 (EMD Millipore Corporation; 1:250), rabbit anti-caspase Dcp1 (Cell Signaling; 1:100) and rabbit anti-pJNK (Promega, 1:200). The secondary antibodies used were as follows: goat anti-mouse Alexa-488, Cy3 and Cy5 (Life Technologies, 1:200) goat anti-rabbit Cy3 and Cy5 (Life Technologies, 1:200) and goat anti-rat Cy3 (Life Technologies, 1:200). F-actin was visualized by means of Rhodamine Phalloidin (Molecular Probes, 1:50). DNA was marked with Hoechst (Molecular Probes, 1:1000).
Confocal images were acquired with a Leica SP5-MP-AOBS or a Zeiss LSM 880 microscope, equipped with a Plan-Apochromat 20× oil objective (NA 0.7), 40× oil objective (NA 1.4) and 63× oil objective (NA 1.4).
In situ hybridization was performed using standard procedures. A digoxygenin-UTP (Boerhringer-Mannheim, Mannheim, Germany)-labeled PVRAP anti-sense RNA probe was generated using the plasmid cDNA EST RE08107 (DGRC).