Anti rabbit alexa 488 secondary antibody

After decapitation and removal of proboscis, heads of 20-day-old GMR-Gal4-driven Aβ flies were fixed for 3 h in 4 % paraformaldehyde in PBS and then incubated overnight in 25 % sucrose in PBS. Heads were frozen in Tissue-Tek O.C.T. (Sakura Finetek) and kept at −80 °C until use. Immunofluorescence was performed on 16 µm cryosections using an anti-Aβ_1–40 mAb antibody (D8Q71, 1/200, Cell Signaling) followed by anti-rabbit Alexa-488 secondary antibody (1/250, Invitrogen), both being diluted in PBS with 0.1 % Triton and 5 % non-fat dry milk. An incubation step of 3 min in 70 % formic acid was included prior to blocking to unmask antigens. Stained sections were mounted using Vectashield with DAPI (Vector) and analysed with a Leica DMI4000B/DFC 340FX inverted microscope. Quantification of Aβ₄₀ deposits was performed using ImageJ from at least 6 flies per genotype.

Tumor masses were fixed in 10% neutral buffered formalin at RT for 24 h and immersed in 30% sucrose/PBS at 4 °C twice, until they were sunk. They were embedded in OCT and stored at − 80 °C. Sections were cut at 10 μm and cells were permeabilized with 0.5% Triton X-100 and blocked in 1% BSA/PBS. Samples were then immune-stained overnight (ON) at 4 °C using a rabbit anti-53BP1 antibody (Novus Biologicals, Centennial, CO, USA). After washes in 1% Bovine Serum Albumin (BSA) dissolved in PBS, samples were incubated with the anti-rabbit Alexa 488 secondary antibody (Invitrogen) for 1 h at 37 °C. Finally, slides were washed in 1% BSA/PBS, counterstained with 4,6-diamidino-2-phenylindole (DAPI; Sigma Aldrich, St. Louis, MO) and analyzed by fluorescence microscopy using an Axio-Imager Z2 microscope equipped with a coupled charged device (CCD) camera (Zeiss, Jena, Germany). The frequency of DNA damage marker foci and colocalization dots per cell were scored in 100 nuclei in at least two independent experiments.