Zebrafish Assays for Identifying Potential Muscular Dystrophy Drug Candidates - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 233

18.2.10 Reactive Oxygen Species Staining Using
H 2 DCFDA Respiratory Burst Assay
ROS production was quantitated in live 3dpf zebrafish using the method described
by Hermann et al. (2004). Single zebrafish were deposited into each well of black
96-well microplates (Corning Life Sciences, Lowell, MA) in 100 mL of fish water.
One hundred microliters of 2 0 ,7 0 -dihydrodichlorofluorescein diacetate (H 2 DCFDA,
Invitrogen) (1 mg/mL) was then added to wells containing zebrafish. After
incubating at 28 C for 1 h, animals were placed on slides and exposed to UV
light for 30 s; fluorescence images were then captured using the same gain and
exposure time.
18.2.11 Motility Assay
To quantitate zebrafish movement, we used the VideoTrack system (ViewPoint Life
Sciences, Lyons, France). The system uses infrared light to view zebrafish in
microwells and to continuously monitor motion. Six dpf zebrafish were placed in
24-well microplates, one animal per well in 500 mL of fish water. Total distance ( D )
traveled by individual zebrafish was recorded for 60 min in alternating 10min light
and dark photoperiods.
18.2.12 Histology
Histology was performed following standard procedures. Briefly, zebrafishwere fixed
in 4%PFA in PBS overnight at 4 C, and embedded in JB-4. Cross and sagittal sections
were prepared and stained in hematoxylin-eosin (H&E) for examination.
18.2.13 Drug Administration
We assessed effects of five drugs: prednisone, EGCG, MG132, TSA, and dantrolene.
Twenty-four hpf MD zebrafish were treated with three compound concentrations (1,
10, and 100 mM) continuously for 48 h. At 3dpf, drug treatment was terminated and
zebrafish were processed to assess myotome length and ROS level. Ten embryos were
used for each condition. 0.1% dimethyl sulfoxide (DMSO) was used as carrier
solvent. To ensure dystroglycan KD specificity, KD control zebrafishwere included in
each experiment.
18.2.14 Fluorescence Image Analyses
Fluorescence intensity in images after ROS staining was quantified by applying
a constant threshold value to ROI (tail region posterior to the anal pore); histograms
of highlighted areas were quantitated using the Adobe Photoshop measurement
function.