Fv10 asw 4.2 viewer

To access the proliferation of chondrocytes, EdU staining assay was utilized using BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 555 (C0075S; Beyotime). Images were captured with a laser-scanning confocal microscope (OLYMPUS FV1000) using the FV10-ASW 4.2 Viewer (OLYMPUS), then analyzed using ImageJ software. Five random visions per sample were chosen to capture the images.

Enucleated mouse eyes were fixed in 1% paraformaldehyde at 4 °C overnight followed by either storage at 4 °C in 50% glycerol and 50% PBS (v/v) for wholemount staining or frozen at −20°C in optimal cutting temperature embedding compound (Electron Microscopy Sciences) and cut into 6-μm thick cryosections for immunofluorescent staining. Nonspecific binding was blocked with 10% heat-inactivated goat serum or 1% bovine serum albumin and anti-mouse CD16/CD32 Fcγ III/II. Primary antibodies were incubated overnight at 4 °C followed by secondary antibodies and DAPI (Invitrogen) incubation for 2 h at room temperature. Images were acquired on a confocal microscope with a ×40 or ×60 oil objective (Olympus FluoView) and analyzed on FV10-ASW4.2 Viewer (Olympus). Antibodies used are shown in Supplementary Table 1.

Immunostaining was performed according to the standard protocol.
For immunofluorescence analysis of the cartilage, frozen sections were incubated with antibodies specific for YAP (1:100, 14074; Cell Signaling), TAZ (1:100, 83669; Cell Signaling), MST1 (1:100, 14946; Cell Signaling), Collagen II (1:100, MA5-13026; ThermoFisher), and MMP13 (1:100, 18165-1-AP; proteintech) overnight at 4°C and then incubated with Cy3-conjugated secondary antibody (1:1000, BA1031, BA1032; BOSTER) for 1 h.
For immunofluorescence analysis of the primary chondrocytes, cells were fixed with 4% paraformaldehyde for 10 min, then incubated in 0.3% Triton X-100 for 15 min, blocked by blocking buffer (1% bovine serum albumin in PBS) for 15 min, and incubated with antibodies specific for YAP (1:100, 14074; Cell Signaling) and Collagen II (1:100, MA5-13026; ThermoFisher) and were then incubated with Cy3-conjugated secondary antibody (1:1000, BA1031, BA1032; BOSTER) for 1 h.
Images were captured with a laser-scanning confocal microscope (OLYMPUS FV1000) using the FV10-ASW 4.2 Viewer (OLYMPUS) and analyzed using ImageJ software.

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).

IFMs were prepared from the adult thoraxes dissected in relaxing buffer (0.1 M KCl, 20 mM Tris-HCl, pH = 7.2, 1 mM MgCl₂, 1 mM EDTA) [34 (link)]. The specimens were fixed in 4% paraformaldehyde for 30 min. After washing with 0.1% PBST (1× PBS, 0.1% TritonX-100) and subsequent blocking in 10% normal goat serum, the primary antibody diluted with the blocking solution was added and incubated at 4 °C overnight. The following primary antibodies were used: anti-Atg8 antibody (#ab109364, Abcam, Cambridge, UK) (dilution 1/400), anti-Ref(2)P antibody (#ab178440, Abcam) (1/500), anti-ATP5A antibody (#ab14748, Abcam) (1/400), and anti-Cleaved Drosophila Dcp-1 antibody (#9578, Cell Signaling Technology, Inc., Danvers, MA, USA) (1/100).
After washing IFM samples with 0.1% PBST, Alexa fluorescence dye-conjugated secondary antibodies (Invitrogen, Waltham, MA, USA) were incubated with the samples for 2 h. For the visualization of F-actin, Alexa Fluor 488-conjugated phalloidin (#A12379, Invitrogen) was added simultaneously. After washing with 0.1% PBST several times, the specimens were embedded with VECTASHIELD Mounting Medium (Vector Laboratories, Burlingame, CA, USA) and observed using a laser scanning confocal microscope (FV10i, Olympus, Tokyo, Japan). Images were acquired at 512 × 512 pixel size. For image processing, FV10-ASW 4.2 Viewer (Olympus) was used.

The mouse eyes were fixed in 4% paraformaldehyde overnight and embedded in paraffin. After dewaxing, rehydration, heat-induced epitope retrieval and blocking with 10% heat-inactivated goat serum, sections were incubated with primary antibodies to myocilin, CHI3L1 (R and D Systems), collagen IV, Ki67(Abcam), AQP1(Santa Cruz) overnight at 4°C. After three washes with PBS, corresponding fluorescent secondary antibodies and 4′,6-diamidino-2-phenylindole (DAPI) were applied to the sections for 1 hr. After five washes, slides were mounted and imaged using a confocal microscope (Olympus IX81) and analyzed on FV10-ASW4.2 Viewer (Olympus). For measuring TM cellularity, primary antibody against collagen IV, together with phase-contrast images, were used to define the TM region in the sections. Cell nuclei stained with DAPI within the TM region were counted under FV10-ASW4.2 Viewer. Images of at least 10 fields per group were photographed, and the number of cell nuclei per field was counted and averaged.