Ix70 confocal laser scanning microscope

Twelve embryos from the uninjected group, 11 from the control-MO injected group, and 14 from the Snrnp200-MO injected group were harvested and cryopreserved per standard procedures. They were fixed in 4% paraformaldehyde, incubated with 30% sucrose, embedded with optimal cutting temperature solution, and frozen in liquid nitrogen for sectioning at 5 μm using a Leica CM1900 cryostat (Leica, Wetzlar, Germany). Rod and cone photoreceptors were further visualized through immunofluorescence staining as indicated previously [12 (link)]. Briefly, cryosections were incubated with designated primary antibodies, including antirhodopsin (Mouse, 1 : 250; Abcam, Cambridge, UK) and zpr-1 antibodies (Mouse, 1 : 250; ZRIC, USA), to label rod and cone photoreceptors, respectively. The cryosections were then treated with fluorescence-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA) for another 1 hr at room temperature and finally counterstained by 4′,6-diamidino-2-phenylindole (DAPI; Sigma, USA) for cell nuclei staining. Images were taken with an Olympus IX70 confocal laser-scanning microscope (Olympus, Tokyo, Japan).

The experiment was performed using a previously described protocol [30 (link)]. Sterile coverslips coated with poly-l-lysine (BD Biosciences) were washed with PBS and incubated with 0.5% glutaraldehyde for 15 min in room temperature. Coverslips were washed with PBS and coated with FITC-conjugated gelatine (Invitrogen) for 10 min. After washing with PBS, coverslips were incubated with sodium borohydride for 1 min and washed again with PBS. To determine the proteolytic activity, cells were seeded onto prepared coverslips (5 × 10⁴ cells) and grown with apelin peptides (100 nM) or ML221 (100 nM) for 24 h. Cells were fixed with 4% formaldehyde and stained with Phalloidin Alexa Fluor^® 568 for filamentous actin detection. Images were collected using an Olympus IX70 confocal laser scanning microscope (Olympus, Tokyo, Japan) with FLUOVIEW FV1000 software (Olympus, Tokio, Japan). Images were analysed through quantification of darker areas lacking fluorescence, which reflected increased activity of ECM degrading proteases using ImageJ software [28 (link)]. The results were calculated as a percent of cells digesting gelatine and then presented as a percent of control. The experiments were performed three times.