Dp71 ccd

Hearts were harvested and fixed in 10% buffered formalin solution for 60 min at room temperature and then for 24 h at 4°C. The specimens were paraffin-embedded, cut into 5 μm thick sections and stained with hematoxylin and eosin (HE). Images were acquired using a light microscope (Nikon/80i, Japan) and a digital camera (DP71CCD, Olympus, Japan).

After the adult zebrafish had been exposed to the alcohol, their eyes and brain tissues were harvested and immediately fixed in 4% paraformaldehyde, equilibrated in 30% sucrose/PBS overnight and embedded in OCT. Twelve-mm-thick sections were mounted on gelatin-coated slides and air dried at 37°C for at least 2 h. The tissue sections were rehydrated with PBS, blocked with 20% NGS and 2% BSA in 0.3% PBS/Triton X-1 00 (PBST) for 1 hr, and incubated with primary antibodies overnight at 4°C. The following primary antibodies and concentrations were used: mouse monoclonal antibody Zpr-1 (1:200, University of Oregon) for labelling cones and mouse monoclonal anti-tyrosine hydroxylase (1:400, Millipore, Billerica, MA) for labelling DA cells. The interpretation of the neuroanatomy follows the adult zebrafish brain atlas [49 ] Immunoreactions were detected using Cy3-labelled goat anti-mouse IgG diluted to 1:400 (Millipore), and the sections were counterstained with a 1:1000 dilution of 4’, 6-diamidino-2-phenylindole (DAPI) (Sigma) to label the nuclei. The slides were examined with an Olympus BX51 light microscope (Olympus, Tokyo, Japan). The images were obtained by an Olympus CCD DP71 (Olympus) and processed using Adobe Photoshop CS (Adobe Systems, San Jose, CA).

Epifluorescence and light microscopy employed an Olympus BX60 equipped with CCD DP71 (Olympus, Tokyo, Japan). Regarding total cell counts, cells in cultures were stained using SYBR Green I solution (Life Technologies, Carlsbad, CA, USA) for 10 min, and filtered using a 0.2-μm-pore polycarbonate membrane (Merck, Burlington, MA, USA). Under the microscope, at least ten randomly selected fields containing more than 50 cells per field were used for quantification; the numbers of cells were counted manually.

After the adult zebrafish had been exposed to the alcohol, their eyes and brain tissues were harvested and immediately fixed in 4% paraformaldehyde. The tissues were routinely processed for paraffin embedding, and 8-mm-thick sections were cut and mounted onto glass slides. The tissue samples were stained with hematoxylin and eosin, and the sections were evaluated and photographed using an Olympus BX51 light microscope equipped with an Olympus CCD DP71 (Olympus).