Coolsnap cf

Cell constructs (100:0–95:5) were cultured for 7 days, fixed with 4% paraformaldehyde and embedded in paraffin, which was then cut into 5-μm thick sections. For histological evaluation, sections of the cell construct were stained with hematoxylin and an aqueous eosin Y solution. Cells were counted by means of a grid (300 × 200 μm) under a light microscope in 4 independent specimens. For immunofluorescent staining, the sections were deparaffinized, then incubated in phosphate buffered saline (PBS) containing 0.1% Triton-X and 1% bovine serum albumin for 20 min. After being washed twice, the sections were incubated with antibodies against CD31 (anti-CD31; 1/50, DAKO, Carpinteria, CA), vascular endothelial growth factor (anti-VEGF; 1/200, Merck Millipore, Billerica, MA), or hepatocyte growth factor (anti-HGF; 1/100, Santa Cruz Biotechnology, Santa Cruz, CA) for 40 min, then incubated with a secondary antibody conjugated with Alexa Fluor 488 (Invitrogen, Carlsbad, CA) for 40 min, followed by nuclear staining with Hoechst33342 (Invitrogen). The stained sections were observed using a fluorescence microscope with a CCD camera (TE2000; Coolsnap cf, Nikon).

Images of zebrafish embryos at 48 hpf were obtained by the stereomicroscope Nikon SMZ1500n with a digital camera CoolSNAP-cf. Adult male fishes (10-months old) of F1 generation were anesthetized, weighted and then photographed by a camera. Eye area and body length were calculated by ImageJ software (RRID:SCR_003070).