Mouse anti diap1

Third-instar larvae were dissected in ice-cold phosphate-buffered saline (PBS). Collected tissues were fixed at 4% paraformaldehyde in PBS for 20 min on ice. After washing twice with PBS, fixed tissues were blocked in 5% normal goat serum/PBT (PBS + 0.3% Triton-X100) for 2–4 h at 4 °C. Samples were incubated with primary antibodies in 5% NGS/PBT at 4 °C overnight. The following antibodies were used for staining: rat anti-Ciao1 (1:100), mouse anti-GFP (1:100) (ab1218, Abcam), sheep anti-GFP (1:100) (4745-1051, BioRad), mouse anti-Elav (1:50) (from K.O. Cho), rabbit anti-BarH1 (1:100) (from J.K. Kang), rabbit anti-PH3 (1:200) (06-570, Millipore), rabbit anti-CycE (1:100) (sc-33748, Santa Cruz), rabbit anti-Cleaved Dcp-1 (Asp216) (9578, Cell Signaling), and mouse anti-Diap1 (1:100) (from B. Hay). After washing three times with PBT, secondary antibodies conjugated with FITC (1:100), Cy3 (1:600), or Cy5 (1:500) (Alexa Fluor, Molecular Probes) were incubated for at least 2 h at room temperature. After washing four times with PBT, Vectashield with DAPI (H-1200, Vector Laboratories) was used to mount the prepared samples. Fluorescent images were acquired using Carl Zeiss LSM710 confocal microscope and ZEN software.

To examine the effect of Tn in DEOM histolysis, white prepupae at 0 h were collected and aged until 8 h APF, 12 h APF, or 24 APF. Muscle preparations were dissected, fixed with 4% formaldehyde in phosphate-buffered saline (PBS) for 30 min and immunostained as indicated^{75 (link)}. To analyze Tn function in salivary gland and midgut histolysis, wandering L3 larvae were dissected, fixed with 4% formaldehyde in PBS, and stained with DAPI (4′,6-diamidino-2-phenylindole) and/or phalloidin. The following primary antibodies were used: rabbit anti-Caspase-3 (1:100, Cell Signaling Technology, Danvers, MA,USA), mouse anti-DIAP1 (1:200, B. Hay)^{76 (link)}, guinea pig anti-p35 (1:10, P. Meier)^{40 (link)}, and guinea pig anti-Tn^{48 (link)}. Secondary antibodies used for fluorescent immunolabeling were Alexa Fluor anti-mouse 488, Alexa Fluor anti-rabbit 488, Alexa Fluor anti-mouse 594, and Alexa Fluor anti-guinea pig 488 (1:400, Molecular Probes, Eugene, OR, USA). Phalloidin 488 and 594 were used for F-actin labeling (1:400, Molecular Probes, Eugene, OR, USA). Immunostained preparations were imaged on a Zeiss LSM 700, processed using the Zeiss Zen software and assembled into figures in Photoshop Elements.