Anti cleaved drosophila dcp 1 antibody

IFMs were prepared from the adult thoraxes dissected in relaxing buffer (0.1 M KCl, 20 mM Tris-HCl, pH = 7.2, 1 mM MgCl₂, 1 mM EDTA) [34 (link)]. The specimens were fixed in 4% paraformaldehyde for 30 min. After washing with 0.1% PBST (1× PBS, 0.1% TritonX-100) and subsequent blocking in 10% normal goat serum, the primary antibody diluted with the blocking solution was added and incubated at 4 °C overnight. The following primary antibodies were used: anti-Atg8 antibody (#ab109364, Abcam, Cambridge, UK) (dilution 1/400), anti-Ref(2)P antibody (#ab178440, Abcam) (1/500), anti-ATP5A antibody (#ab14748, Abcam) (1/400), and anti-Cleaved Drosophila Dcp-1 antibody (#9578, Cell Signaling Technology, Inc., Danvers, MA, USA) (1/100).
After washing IFM samples with 0.1% PBST, Alexa fluorescence dye-conjugated secondary antibodies (Invitrogen, Waltham, MA, USA) were incubated with the samples for 2 h. For the visualization of F-actin, Alexa Fluor 488-conjugated phalloidin (#A12379, Invitrogen) was added simultaneously. After washing with 0.1% PBST several times, the specimens were embedded with VECTASHIELD Mounting Medium (Vector Laboratories, Burlingame, CA, USA) and observed using a laser scanning confocal microscope (FV10i, Olympus, Tokyo, Japan). Images were acquired at 512 × 512 pixel size. For image processing, FV10-ASW 4.2 Viewer (Olympus) was used.