Anti cleaved dcp 1

Leg discs from prepupae (from 0h to 4h after puparium formation depending on experiments) are dissected in PBS 1x. Tissue are fixed by paraformaldehyde 4% diluted in PBS 1x during 20 minutes. Then the samples are washed and saturated in PBS 1x, 0.3% triton x-100 and BSA 1% (BBT). Next, the samples are incubated overnight at 4°C with primary antibodies diluted in BBT. Samples are washed for 1h in BBT before a 2h incubation at room temperature with secondary antibodies diluted in BBT. Finally, samples are washed with PBS 1x, 0.3% Triton x-100 for 1h and mounted in Vectashields containing DAPI (Vector Laboratories). A 120-μm-deep spacer (Secure-Seal™ from Sigma-Aldrich) is placed in between the glass slide and the coverslip to preserve morphology of the tissues.
Primary antibodies from Developmental Studies Hybridoma Bank (DSHB) are klarsicht-C antibody (9C10-s, mouse, 1:50), Lamin Dm0 (ADL195-s, mouse, 1:50) and E-Cadherin antibody (DCAD2, rat, 1:50). Anti-cleaved Dcp-1 (#9578, rabbit, 1:200) was obtained from Cell Signaling Technologies. Secondary antibodies (Alexa488 and 647) are purchased from Interchim and diluted at 1:200 or 1:100 respectively. Phalloidine-Rhodamine (Fischer Scientific) used to stain F-actin is diluted at 1:200.

Flies were dissected in Grace's media and ovaries were fixed and stained as described previously (Tanner et al., 2011 (link)). Samples were mounted in VectaShield with DAPI (Vector Labs). Primary antibodies used were: anti-cleaved-Dcp-1 (1:100, Cell Signaling Technology), anti-Dlg [1:100, Developmental Studies Hybridoma Bank (DSHB)], anti-αPS3 (1:300, Shigeo Hayashi, or 1:1000), anti-βPS (1:10, DSHB), anti-Drpr (1:50, DSHB 5D14), anti-β-Gal (1:400, Promega), anti-aPKC (1:1000, Santa Cruz Biotechnology, Inc.), anti-Dynein (1:3, DSHB 2C11), anti-Kinesin (1:100, Cytoskeleton, Inc.), anti-Crumbs (1:25, DSHB Cq4, protocol from Tanentzapf et al., 2000 (link)) and anti-Talin (mixture of A22A and E16B, 1:50 each, DSHB). The anti-αPS3 antibody was made using the peptide sequence utilized for the original antibody (Wada et al., 2007 (link)) and generated by YenZym (San Francisco, CA). The serum was affinity-purified twice before use. It shows the same expression pattern as the original antibody from the Hayashi lab. Secondary antibodies used were goat-anti-rabbit Cy3, goat-anti-mouse Cy3, goat-anti-mouse Alexa Fluor 647 (Jackson ImmunoResearch), each at 1:100, and goat-anti-rabbit Alexa Fluor 488 (Invitrogen) at 1:200. Egg chambers were imaged on an Olympus FV10i confocal microscope, images were processed using ImageJ and Adobe Photoshop, and figures were made using Adobe Illustrator.

Larval discs were dissected in 1xPBS, fixed 20min in 4% paraformaldehyde at room temperature, rinsed 3x in 1xPBS, then permeabilized for 30min at room temperature (RT) in 1xPBS+0.3% Triton X-100 (1xPBST 0.3%). Samples were then blocked for 1 hour in 1xPBS+10% normal goat serum (NGS), and then incubated in primary antibody 1xPBST 0.1% + 10% NGS overnight at 4°C. The next day samples were washed for 5min 5x in 1xPBST 0.1%, incubated for 45min at RT in secondary antibody in 1xPBST 0.1% + 10% NGS, and washed for 5min 5x in 1xPBST 0.1%. Nuclei were then stained with DAPI/Hoescht for 10min and washed 2x in 1xPBS, before mounting discs in n-propyl gallate (4% w/v in glycerol). Wing discs were imaged on a Nikon A1R HD25 confocal system using 20x and 40x objectives.Primary antibodies: anti-Wg antibody (DSHB, 4D4; 1:50), anti-β-galactosidase (DSHB, 40–1a; 1:250), anti-EcR common (DSHB; 1:100), anti-cleaved DCP-1 (Cell Signaling; 1:100), and anti-Phospho-histone H3 (Cell Signaling; 1:100). Fluorescently labeled secondary antibodies were from Jackson Immunoresearch Laboratories (1:50). Nucleic acid staining was performed by incubating discs for 10 min with Hoescht 3342 (Thermo Fisher Scientific) or DAPI (Invitrogen).