Dmi4000b dfc 340fx inverted microscope

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.

This procedure was based on³⁴ with some modifications. Briefly, embryos were washed in saline solution (0.7% NaCl and 0.03% Triton x-100) and dechorionated with bleach for 3 min. Following fixation in fixative solution (1 vol of heptane and 1 vol of formaldehyde 4%) for 30 min at room temperature, embryos were incubated for one hour at 4 °C in blocking solution (5% BSA in PBT with 0.2% of Triton X-100) and then subsequently incubated overnight at 4 °C with the anti-dTau antibody (1/1000 in PBT with 5% BSA) or with the anti-hTau K9JA antibody (Dako, 1/5000 in PBT with 5% BSA). Alexa Fluor 488 secondary antibody was used at a dilution of 1/200 in PBT with 5% BSA for two hours at 4 °C. Embryos were mounted in Vectashield with DAPI mounting medium and visualised using a LEICA DMI4000B/DFC 340FX inverted microscope and a 10X objective. Flies used for these experiments were kept at 25 °C.

This procedure was based on Kaczynski and Gunawardena36 with some modifications. Briefly, following removal of the chorion and fixation in a 2% formaldehyde solution, embryos from wt and tau KO flies were incubated for one hour in blocking solution (5% BSA in PBT containing 0.2% of Triton X-100) at 4 °C and subsequently incubated overnight at 4 °C with the anti-dTau antibody (1/1000 in PBT with 5% BSA). Alexa Fluor 488 secondary antibody was used at a dilution of 1/200 in PBT with 5% BSA for two hours at 4 °C. Embryos were then mounted in Vectashield with DAPI mounting medium and visualised using a LEICA DMI4000B/DFC 340FX inverted microscope and a 10× objective.