Uplsapo30xs

The VSMCs were incubated on glass-bottom culture dishes (No. 1, Matsunami Glass Industry) coated with 5% fibronectin (F1141, Sigma-Aldrich) in phosphate-buffered saline (PBS) for 60 min. The cells were imaged under an inverted fluorescence microscope (IX71, Olympus) equipped with a birefringence imaging system (Abrio-LS, CRi)^{21 (link),40 (link)} to analyse the retardation in samples. Because the retardation in background areas changed with time, a background image was taken just before imaging a cell. Cell retardation images were processed by subtracting background retardation from cell retardation. The retardation image was captured through an objective lens of 60× (UPLSAPO60XW, NA = 1.20, Olympus; UPLSAPO60XS2, NA = 1.30, Olympus) or 30× (UPLSAPO30XS, NA = 1.05, Olympus) with silicone immersion oil (SIL300CS-30CC, Olympus). DIC images of the cells were also obtained.

3D immunohistological analysis was performed according to our original protocol^{30 (link)}. Pre-fixed, frozen liver samples were prepared by the same protocol as that used for 2D immunohistology. Samples were cut into 200-μm-thick sections using a cryostat microtome. After washing with PBS, the samples were permeabilized with blocking/permeabilization reagent and subjected to primary antibody staining, washing, and secondary antibody staining. Nuclei were counterstained with Hoechst33342 or SYTOX Green (Thermo Fisher). All procedures were performed at 4 °C on a rocking device, and antibody concentrations were the same as those used for 2D immunohistology (Supplementary Table 2). After staining, samples were treated with SeeDB^{44 (link)} overnight. Images were acquired under a confocal microscope (FV-1000 or FV3000; Olympus) with a 30× silicone immersion lens (UPLSAPO30XS; Olympus). 3D images were reconstructed with IMARIS software (Bitplane, Zurich, Switzerland).

Staining and optical clearing of dissected tracheae were performed as described in a previous study (Hirashima and Adachi, 2015 (link)). Briefly, the samples were fixed with 4% PFA in PBS overnight at 4°C. For anti-SOX9 staining, the samples were incubated in 25 mg/mL hyaluronidase (Nacalai Tesque, #18240-36) for 1 h at 37°C, to digest hyaluronic acid. The samples were then blocked in 10% normal goat serum (Abcam, #ab156046) diluted in 0.1% Triton X-100/PBS (PBT) for 3 h at 37°C. The samples were treated with primary antibodies overnight at 4°C, washed in 0.1% PBT, and subsequently incubated in secondary antibodies conjugated to either Alexa Fluor 546 or Alexa Fluor 647 overnight at 4°C. DAPI was used for nuclear counterstaining (Dojindo Molecular Technologies, #D523-10, 1:200 dilution). The samples were mounted with 10 μL of 1% agarose gel onto a glass dish (Greiner Bio-One, #627871) for stable imaging. Then, the samples were immersed in CUBIC-R+ (Tokyo Chemical Industry Co., # T3741) solution for optical clearing. Images were acquired using the confocal laser scanning platform Leica TCS SP8 equipped with the hybrid detector Leica HyD, using a ×40 objective lens (NA = 1.3, WD = 240 μm, HC PL APO CS2, Leica) and the Olympus FluoView FV1000 with a ×30 objective lens (NA = 1.05, WD = 0.8 mm, UPLSAPO30XS, Olympus).

Zebrafish eggs at the one-cell stage were microinjected with 50 ng/µL of GFP-LC3 mRNA, which was synthesized from pcDNA3-GFP-LC3-RFP-LC3ΔG plasmid (Kaizuka et al., 2016 (link)) using a mMESSAGE mMACHINE T7 Transcription Kit (AM1344, Thermo Fisher Scientific) and purified using RNeasy Mini Kit (74104, Qiagen). Embryos were anesthetized with 0.03% tricaine (A5040, Sigma-Aldrich), placed in water on a glass-bottomed dish, and viewed using a confocal microscope (FV1000 IX81; Olympus) with an objective lens (UPLSAPO30XS, Olympus).

Imaging was performed on the Olympus FV1200 confocal microscope with GaAsP sensors. A 30×/1.05 silicone immersion objective (UPLSAPO30XS, Olympus) (Figure 1C–D and Figure 2—figure supplement 1B), or a 60×/1.42 oil immersion objective (PLAPON60XO, Olympus) (Figures 2B–D and 3A, Figure 2—figure supplement 1A and Figure 3—figure supplement 1A) was used for scanning specific regions of interest (ROIs). A final voxel size of the image was 0.17 × 0.17 × 0.76 μm³ (Figure 1C–D), 0.11 × 0.11 × 0.45 μm³ (Figure 2B and Figure 2—figure supplement 1A), 0.21 × 0.21 × 0.43 μm³ (Figure 2C–D), 0.10 × 0.10 × 0.45 μm³ (Figure 3A), 0.51 × 0.51 × 0.68 μm³ (Figure 2—figure supplement 1B), and 0.79 × 0.79 × 0.37 μm³ (Figure 3—figure supplement 1A), respectively. Confocal stacks were analyzed with the open-source software ImageJ (National Institute of Health) and Fiji (Schindelin et al., 2012 (link)). Where appropriate, 2D/3D image deconvolution was applied using Diffraction PSF 3D and Parallel Iterative Deconvolution plugins in ImageJ.