Fv1200 confocal microscope system

To prepare samples for imaging, samples were incubated in a refractive index matching solution (such as RapiClear or Histodenz) for 1–24 hours depending on sample size. Samples were mounted as previously described^{8 (link), 16 (link)}. Samples were imaged using an Olympus FV1200 confocal microscope system running Fluoview software, using a 10x, 0.6NA water immersion Olympus objective at 5 μm z-step resolution. Single photon excitation was used at the indicated wavelengths. Entire samples were obtained by mosaic tiling during imaging, reconstructed using default microscope software, and viewed in Imaris 8 (Bitplane).

HT22 cells plated on coverslips were fixed with 4% paraformaldehyde at room temperature for 15 min and sealed with 10% donkey serum at room temperature for 1 h. Afterward, the cells were incubated overnight at 4°C with the following primary antibodies: anti-PSD95 (ab18258, Abcam, United States) or anti-p-CaMKII (ab5683, Abcam, United States), incubated with donkey anti-rabbit fluorescent secondary antibody (A21206, Invitrogen, United States) for 2 h at room temperature in the dark, and counterstained with DAPI (Sigma, United States) for 10 min. Fluorescence images were obtained by using an inverted Olympus FV1200 confocal microscope system. The relative mean fluorescence intensity was measured using Image-Pro Plus software (Media Cybernetics).

To obtain the images of embryos, the pigmentation of the embryos was suppressed by the addition of 1-phenyl-2-thiourea (PTU) (Sigma-Aldrich, St. Louis, MO) into breeding E3 media. Embryos were dechorionated and mounted in 1% low-melting agarose dissolved in E3 medium. Confocal images of 2.0 μm steps were taken with a FV1200 confocal microscope system (Olympus, Tokyo, Japan) equipped with a water immersion 20x lens (XLUMPlanFL, 1.0 NA, Olympus). Images were processed with a FV10-ASW 4.2 viewer (Olympus). The distance between the hand2-positive region of the ALPM and the PLPM was measured using DP2-BSW software (Olympus). Cell-tracking data containing nuclei positions were analyzed using Imaris8.4.1 software (Bitplane, Zurich, Switzerland).