Rhodamine phalloidine

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.

To limit the intrinsic variability in the apoptotic pattern, imaginal leg discs were dissected 2 h after pupal formation in PBS 1X, then washed in PBS 1x, 0.3% triton x-100 and BSA 1% (BBT), then incubated overnight at 4°C with primary antibodies diluted in BBT. After the primary antibody, the samples were washed for 1 h in BBT, incubated for 2 h at room temperature with secondary antibodies diluted in BBT, washed with PBS 1x, 0.3% Triton X-100 for 1 h, and mounted in Vectashield (Vector Laboratories), using a 120-μm deep spacer (Secure-SealTM from Sigma-Aldrich) to preserve tissue morphology.
Anti-Dcp1 (Cell Signaling Technologies) and anti-Dpn (ab195172; Abcam) were both used at a dilution of 1/100. Phalloidine–Rhodamine (Thermo Fisher Scientific) used to stain F-actin was diluted at 1:200.