Uplanapo 20

Laser ablation was conducted as described previously^{73 (link)}, using an IX81 inverted microscope (Olympus) equipped with a spinning disk confocal unit (CSU-X1; Yokogawa Electric Corporation) and iXon3 897 EM-CCD camera (Andor), and controlled with IQ2 software (Andor). A dry objective lens (UPlanAPO ×20, N.A. 0.7; Olympus) was used. An N2 Micropoint laser (16 Hz, 365 nm, 0.3 μW; Photonic Instruments) was focused on the plasma membrane at a cell–cell junction labeled with Stargazin-mEGFP-iLID or mEGFP-KRasCT. Time lapse images were acquired every 3.08 s before and after the course of the laser ablation. Before ablation, the embryos in the incubator were illuminated from overhead by blue light for 20 min. The vitelline membrane was manually removed before imaging to clearly visualize the cell–cell junctions.

The coverslip, which was bound with target miRNAs and fluorescent probes, was placed on a fluorescence microscope (IX70, Olympus, Tokyo, Japan). 6-FAM-labeled C-probe, Alexa532-labeled D-probe and Alexa647-labeled D-probe were observed in the epi-illumination mode with a 50-W metal halide light source (U-LH50MH, Olympus, Tokyo, Japan) using the appropriate excitation band pass filter (BP460-490, Olympus, Tokyo, Japan for 6-FAM; D520/40M, Chroma Technology Corp., Bellows Falls, VT, USA for Alexa532; Semrock – FF01-628/40 BrightLine, IDEX Corp., Lake Forest, IL, USA for Alexa647). The emission light from the probes was collected by an objective lens (UPLANAPO 20×, N.A.0.70, Olympus, Tokyo, Japan) and captured by an electron-multiplying charge-coupled device (EM-CCD) camera (iXon3, Andor Technology Japan, Tokyo, Japan) using the appropriate emission band pass filter (HQ535/50 M, Chroma Technology Corp. for 6-FAM; Semrock - FF01-593/40 for Alexa532; Semrock – FF01-692/40 BrightLine, IDEX Corp. for Alexa647). Circular fluorescent probe spots (Figure 1C) in recorded images were enclosed by ellipsoidal regions of interest (ROIs) and the fluorescence intensities in ROIs were analyzed using NIH ImageJ software. Background intensities without C-probe were used for subtraction from the raw data.