Cytoprotective Activities of Water-Soluble Fullerenes in Zebrafish Models - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 256

reconstruction of 3D objects and analyzes Z-stacks. Images were analyzed with
AxioVision software Rel 4.0 (Carl Zeiss Microimaging Inc.), the Adobe Photoshop
6.0 computer program (Adobe, San Jose, CA), and NIH image software (Bethesda,
MD). Patterns and intensity of staining were recorded and quantitated.

19.2.6.5 AcridineOrange Staining At 48, 72, and 120hpf, five embryos were
immersed in 0.5 g/mL acridine orange (acridinium chloride hemi(zinc chloride)) in

PBS for 60min and rinsed thoroughly twice in 10mL of fresh fish water. Stained
embryos were anesthetized with MESAB and mounted in methylcellulose in a
depression slide for observation using fluorescent microscopy. Effects of water-
soluble fullerenes on apoptosis in the hatching glands, retina, lateral neuromasts,
and olfactory pits were examined.

m

19.2.7 Determination of Dopaminergic
CNS Neuroprotective Activity Against
6-Hydroxydopamine-Induced Apoptosis

19.2.7.1 Treatment of Embryos with 1-12 Two-day zebrafish were treated
with 1% DMSO for vehicle control and treated with 1% DMSO

M 6-OHDA
for DA-loss control. For compound testing, 2-day embryos were exposed to a mixture
of 250

þ 250 m

m M 6-OHDA and fullerenes for 72 h.

19.2.7.2 Antibody Staining Embryos were fixed in 4% paraformaldehyde
overnight at 4 C. Fixed embryos were permeabilized with cold acetone at

20 C for
20min and rehydrated in stepwise descending ethanol/PBS solutions (95, 79, 50, 25,
and 0% ethanol/PBSmix, 10min for each step). Embryos were then stained with anti-
tyrosine hydroxylase antibody (mouse anti-human, Sigma, St. Louis, MO) at 4 C
overnight. The next day, samples were washed with PBS-T (0.1% Tween 20),
incubated with secondary antibody (goat anti-mouse), and color was developed
using ABC reagent (Vector Labs, Burlingame, CA) according to manufacturer's
instructions. Stained embryos were further flat-mounted on a glass slide and five
randomly selected embryos were examined for DA neuron loss.

19.2.7.3 Light Microscopy and Image Analyses All microscopy studies
were performed using a Zeiss light microscope (Carl Zeiss Microimaging Inc.)
equipped with a SPOT camera (Diagnostic Instruments, Sterling, MI). The patterns
and the intensity of staining were recorded and quantified.

19.2.7.4 Fluorescence Microscopy All fluorescence microscopy studies
were performed using a Nikon Eclipse E600 fluorescence microscope (Nikon
Inc., Melville, NY) equipped with a 200W mercury/xenon lamp, a green FITC
filter (excitation: 488 nm, emission: 515 nm), and a C5985 chilled CCD camera