Rabbit anti phospho drosophila akt ser505

Third-instar larvae were dissected in ice-cold phosphate-buffered saline (PBS). Collected tissues were fixed at 4% paraformaldehyde in PBS for 15 min. After washing twice with PBS, fixed tissues were blocked in 5% normal goat serum/PBT (PBS + 0.3% Triton X-100) for 30 min at room temperature. Samples were incubated with primary antibodies in 5% NGS/PBT at 4 °C for overnight. Following antibodies were used: rabbit anti-CCT4 (1:2000) (this study), rabbit anti-Dlg (1:500) (Kyung-Ok Cho, KAIST), mouse anti-Dlg (1:100) (DSHB), sheep anti-GFP (1:100) (Bio-Rad, 4745-1051), rabbit anti-Cleaved Caspase-3 (Asp175) (1:100) (Cell Signaling, 9661), rabbit anti-phospho-Drosophila Akt (Ser505) (1:100) (Cell Signaling, 4054). After washing three times with PBT, secondary antibody conjugated with FITC (1:100), Cy3 (1:600), or Cy5 (1:500) (Alexa Fluor, Molecular Probes) were incubated for 1 h at room temperature. For Phalloidin staining, Alexa Fluor 488 Phalloidin (Molecular Probes A12379) was incubated for 1 h at room temperature. After washing three times with PBT, Vectashield (Vector Laboratories) antifade reagent was used for mounting prepared samples. A Carl Zeiss LSM710 confocal microscope was used to acquire fluorescent images.