Wandering third instar larvae were collected and eye imaginal discs dissected. For clone induction, larvae were given a heat shock at 37°C 48h or 72h before dissection. For pupal dissections, white prepupae (0hr) were collected and maintained at 25°C for 40h. Dissections were performed in chilled PBS, samples were fixed for 30min in formaldehyde (4% v/v in PBS) and permeabilized with PBT 0,4%Triton.
The primary antibodies and fluorescent reagents used in this study are listed in theKey Resources Table . TUNEL staining (Roche) was performed according to the supplier’s protocol and modified as previously (Lolo et al., 2012 (link)). For detection of protein inclusions, brains were fixed and permeabilized as described above and incubated for 1h30min with the proteostat aggresome dye (Enzo Life Sciences) before mounting. For the necrosis assay, brains were dissected in PBS 1X and incubated for 30min at room temperature with 10 μg/ml propidium iodide (PI) (Sigma-Aldrich) in Schneider medium, following by washing and standard fixation ((Liu et al., 2014 (link), Yang et al., 2013 (link))). Samples were mounted in Vectashield (Vectorlab) and imaged on a Leica confocal SP5 or a Zeiss LSM 880 using a 20X dry objective or a 40X oil objective.
The primary antibodies and fluorescent reagents used in this study are listed in the