Imaging of Cell Death in Imaginal Discs

At least 20 imaginal discs per experiment were dissected from late third instar larvae and pupal retinae from 42-h-old pupae. They were fixed and stained using standard protocols (Fogarty and Bergmann, 2014 (link)). TUNEL was performed using a TUNEL assay kit (Roche Life Sciences) according to the manufacturer's instructions. Antibodies to the following primary antigens were used: ELAV [rat; 1:50; Developmental Studies Hybridoma Bank (DSHB)]; Cut (mouse; 1:50; DSHB); Dlg (rabbit; 1:100; from Kwang-Wook Choi, Korea Advanced Institute of Science and Technology, Daejeon, South Korea); MBNL1 (rabbit; 1:2000; from Charles Thornton, University of Rochester Medical Center, Rochester, NY, USA); cleaved Caspase 3 (rabbit; 1:200; Cell Signaling Technology, 9661); and eIF2α (P-Ser51) (rabbit; 1:100; Cell Signaling Technology, 3597). Secondary antibodies were donkey Fab fragments from Jackson ImmunoResearch (715-166-151, 711-096-152, 712-606-153, 711-166-152; all at 1:600). Nuclei were visualized by Hoechst and DAPI staining. Fluorescent images were taken with an Olympus Optical FV500 confocal microscope.

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Yenigun V.B., Sirito M., Amcheslavky A., Czernuszewicz T., Colonques-Bellmunt J., García-Alcover I., Wojciechowska M., Bolduc C., Chen Z., López Castel A., Krahe R, & Bergmann A. (2017). (CCUG)n RNA toxicity in a Drosophila model of myotonic dystrophy type 2 (DM2) activates apoptosis. Disease Models & Mechanisms, 10(8), 993-1003.

Publication 2017

TUNEL assay kit cleaved Caspase 3 donkey Fab fragments

Antibodies Antigens Assay Caspase 3 Confocal optical microscope Dapi Donkey Fab fragments Hybridoma Imaginal discs Larvae Mouse Nuclei Pupae Rabbit Retinae Tunel

Corresponding Organization :

Other organizations : The University of Texas MD Anderson Cancer Center, University of Massachusetts Chan Medical School, Valentia Biopharma (Spain), The University of Texas Health Science Center at Houston

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Variable analysis

independent variables

Late third instar larvae
Pupal retinae from 42-h-old pupae

dependent variables

Cell death (TUNEL assay)
ELAV expression
Cut expression
Dlg expression
MBNL1 expression
Cleaved Caspase 3 expression
EIF2α (P-Ser51) expression

control variables

At least 20 imaginal discs per experiment were dissected
Tissues were fixed and stained using standard protocols
Antibodies were used at specified dilutions
Nuclei were visualized by Hoechst and DAPI staining
Fluorescent images were taken with an Olympus Optical FV500 confocal microscope

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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