Femtojet

GV oocytes and PN embryos were microinjected with Sebox and GFP dsRNA in M2 medium containing 0.2 mM IBMX or in M2 medium alone, respectively. An injection pipette holding dsRNA solution was inserted into the cytoplasm of oocytes or embryos, and 10 pl dsRNA was microinjected with a constant-flow system (Femtojet; Eppendorf, Hamburg, Germany). To achieve the Mll stage, oocytes were cultured in M16 containing 0.2 mM IBMX for 8 h, followed by culture in plain M16 for 16 h in 5% CO₂ at 37°C. Similarly, GFP and Sebox dsRNA-microinjected PN embryos were developed to the 2C stage in M16 medium containing 100 μM EDTA (Sigma-Aldrich).

5xFAD mice and control mice (C57BL/6 x SJL) were injected twenty days before imaging, EGFP- LC3 lentivirus (titer: 5.0 × 10⁶ vector genomes/ml, 5 μl) was injected into the retrosplenial dysgranular cortex (RSD, anteroposterior, −2.0 mm and mediolateral, 0.6 mm from bregma; depth, 1 mm), and the cerebellum cortex (anteroposterior, −7.0 mm and mediolateral, 1.2 mm from bregma; depth, 0.5 mm) of mice under anesthesia with 1.5% isoflurane. Human carboxytetramethylrhodamine (TAMRA)-β-amyloid 1–42, (100 μM, diluted by ACSF, DMSO, 0.1%; 1 μl) was injected into the RSD of mice under anesthesia with 1.5% isoflurane one day before imaging. Virus was delivered via a glass micropipette of 20–50 μm tip diameter made by capillary puller (P-1000 Pipette Puller, Sutter Instrument) under the conditions Heat at 741, pull power at 150, Velocity at 75, Delay at 1000 and Pressure at 400. 5 ul of viral solution was injected at the speed of 1 μl/min into RSD with an injection machine (FemtoJet^®, eppendorf).

The GV oocytes were microinjected with miRNA mimics in M2 medium containing 0.2 mM IBMX. An injection pipette containing the miRNA mimic solution was inserted into the cytoplasm of an oocyte, and 10 pl of 2 μM miRNA mimic, 2 μM miRNA inhibitor or Lin28a small interfering RNA (siRNA; Dharmacon) was microinjected using a constant flow system (Femtojet; Eppendorf). To assess injection damage, oocytes were injected with a negative control miRNA mimic and control siRNA (Dharmacon), which were used as negative controls. To determine the rate of in vitro maturation, oocytes were cultured in M16 medium containing 0.2 mM IBMX for 24 h and then cultured in M16 medium alone for 16 h in 5% CO₂ at 37°C. After the miRNA mimic microinjection experiments, the maturation stage of the oocytes was scored based on the presence of a GV oocyte, a polar body (MII oocyte) or neither a GV nor a polar body (MI oocyte).

All reporter plasmids were integrated into a unique landing site on the third chromosome using the VK00033 strain (60 (link)). PhiC31 was maternally provided using the vas-phiC31 strain (61 (link)). Microinjection was performed as previously described (62 (link)). In brief, 0–1 h embryos were collected and dechorionated with bleach. Aligned embryos were dried with silica gel for ∼7 min and covered with FL-100-1000CS silicone oil (Shin-Etsu Silicone). Subsequently, microinjection was performed using FemtoJet (Eppendorf) and DM IL LED inverted microscope (Leica) equipped with M-152 Micromanipulator (Narishige). Injection mixture typically contains ∼500 ng/μl plasmid DNA, 5 mM KCl, 0.1 mM phosphate buffer, pH 6.8. The mini-white marker was used for screening.

110-day-old male flies were imaged before and after bolus injection of 10 mM EGTA or 100 µM valinomycin (Sigma-Aldrich, St. Louis, MO) containing artificial hemolymph-like solution (AHLS): 113 Na⁺, 5 K⁺, 8.2 Mg²⁺, 2 Ca²⁺, 133 Cl⁻, 5 HEPES, 4 HCO₃⁻, 1 H₂PO₄,⁻, 10 Sucrose, 5 Trehalose, pH 7.5 (in mM). Borosilicate glass pipettes (1 mm OD, 0.75 mm ID, A-M Systems, Sequim, WA) were pulled using a P-1000 puller (Sutter Instruments). Pipette tip diameters of 50–75 µm were created by crushing the taper with forceps and visually confirming their diameter using a microforge (Narishige MF-830). Bolus injections into the abdomen of flies under ice anesthesia were approximately 1000 pL in volume and were made using a Femtojet (Eppendorf, Hamburg, Germany), with the pipette positioned using a manual micromanipulator (World Precision Instruments, Sarasota, FL).