Fv31s sw

Zebrafish embryos were treated with 2 mM EDU (5-ethynyl-2′- deoxyuridine) for 30 min at 4 °C in egg-water. The embryos were transferred to fresh E3 medium for 2 h and fixed in 4% paraformaldehyde at 4 °C overnight. We treated the zebrafish embryos with Cell-Light™ EdU Cell Proliferation Detection (C10310-1, Ribobio, Guangzhou, China), according to the operating protocol, to visualize the proliferating cells. For the triple labeling of EDU, p63, and dlc, the samples were continuously treated in accordance with the standard immunofluorescence procedure and assessed via a confocal microscope (FV31S-SW, Olympus), as mentioned in Section 2.4.
TUNEL staining was performed as previously described [45 (link)]. The apoptosis cells were labeled with the TUNEL BrightRed Apoptosis Detection Kit (Vazyme Biotech, NanJing, China). For double labeling of the TUNEL and atp1a1a.4 and the TUNEL and atp6v1a, as well as triple labeling the TUNEL, p63, and dlc, the samples were treated in accordance with the standard immunofluorescence procedure, and assessed by a confocal microscope (FV31S-SW, Olympus), as mentioned in Section 2.4.

MEFs expressing GFP-actin were incubated to confluency and serum-starved for 24 h before analysis. Fluorescence microscopy was performed using a confocal laser microscope (FV3000; Olympus) with a 100 × oil-immersion objective lens (1.40 NA; Olympus) and captured with FluoView software (FV31S-SW, Olympus). The chamber was maintained at 37 °C and continuously supplied with humidified 5% CO₂. Five pre-bleaching images were acquired at 5-s intervals, and circular regions of interest (ROI) with radii of 2 µm were selected and bleached with an 80% power 488-nm laser for 800 ms. Fluorescence recovery was recorded for 250 s by acquiring images at 5 s intervals. Pre- and post-bleach images were taken with 0.5% power. Fluorescence intensity was analyzed by Fiji and its simFRAP plug-in (https://imagej.nih.gov/ij/plugins/sim-frap/index.html). Briefly, the mean intensity values of the ROIs, total image, and background fluorescence were calculated. After background subtraction, the mean intensity values of ROIs were normalized with those of each total image for each time-point.

In total, 24 h after transfection and 4, 8, 16 h after MG132 treatment, cells were fixed with ice–cold methanol for 7 min. Cells were then blocked with 5% bovine serum albumin for 1 h and subsequently incubated in a humidified chamber with the primary mouse anti-FLAG antibody (1:500, Sigma-Aldrich, 2 h). After primary incubation, the cells were washed three times with PBS and incubated for 1 h with a secondary goat anti-mouse antibody conjugated to AlexaFluor 555 (1:500, Molecular Probes, Invitrogen), protected from light. Cells were then washed three times with PBS and incubated with Hoechst (1:10.000, Merck, Germany) to counterstain nuclei. Primary and secondary antibodies were diluted in 1% bovine serum albumin, Hoechst was diluted in PBS. Coverslips were mounted using 50% glycerol/PBS and analyzed by Confocal microscopy. Confocal microscopy was performed on an Olympus FV3000laser-scanning confocal microscope (Evident Scientific, Hamburg, Germany) equipped with 405 nm and 561 nm laser sources. Sequential confocal images were acquired using a UPLXAPO 40x oil immersion objective (1.40 numerical aperture) with a 1024 × 1024 format, scan speed 8 μs/pixel and z-step size of 0.4 μm. Maximal Intensity Projection (MIP) of each Z-reconstruction was obtained by FV31S-SW (version 2.4.1.198) Olympus software.