Radiation-Induced Toxicity and Radiation Response Modifiers in Zebrafish - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 287

4 mg/mL tricaine methanesulfonate (Sigma) and dorsally positioned on a 3%
methylcellulose gel. Tetramethylrhodamine-labeled, 10 kDa dextran (Molecular
Probes) is injected into the cardiac venous sinus and embryos imaged 1, 5, and
24 h following injection. The average fluorescence emission at 590 nm following
excitation at 570 nm is detected over the cardiac area, and the relative intensity
measured using a Leica microscope (Leica Mikroskopie and Systeme GmbH).
Images are transformed into grayscale and evaluated with NIH ImageJ software as
described (Hentschel et al., 2005).

21.2.5 Assessment of Gastrointestinal Morphology
and Function

The functional and morphological integrity of the developing gastrointestinal system
is assessed in zebrafish embryos using PED6, a fluorescent reporter of phospholipase
A2 (PLA2) activity. PED6 is a fluorogenic substrate for PLA2, which contains a
BODIPY FL dye-labeled acyl chain and a dinitrophenyl quencher group (Farber
et al., 2001). The cleavage of the dye-labeled acyl chain by phospholipase A2 within
cells lining the intestine unquenches the dye and leads to the fluorescent labeling of
the gall bladder and the lumen of the developing gastrointestinal tract. PED6 is added
to zebrafish embryos at a stage when feeding and swallowing has commenced (5 dpf)
followed by imaging the fish at 6dpf with the average fluorescence emission at 540 nm
and excitation at 505 nm. Images are taken using a Leica microscope and analyzed
using ImageJ software.

21.2.6 Ototoxicity Assay

Neuromast development is assessed as described by Harris et al. (2003), using the
fluorescent vital dye 2-[4-(dimethylamino)styryl]-N-ethylpyridinium iodide (DASPEI,
Molecular Probes, Carlsbad, CA). Zebrafish embryos at 5 dpf are exposed to 80Gy.
Neuromast staining was done 24 h later by incubation with 0.005%DASPEI in embryo
mediumfor 15min. Embryos are rinsed oncewith embryomediumand anesthetized for
5min. The fluorescent emission at 515 nm following excitation at 450-490 nm is
detected using a fluorescence microscope (Olympus BX51, Olympus). Neuromast
staining was evaluated as follows: each neuromast on one side of the body is given a
DESPEI score of 0 for no

staining. Values are normalized to the maximum possible score of 54 (27 neuromasts).

þ 2 for normal staining, þ 1 for reduced staining, and þ

21.2.7 Determination of Apoptosis by Acridine
Orange Staining

Six hours after radiation exposure (20 Gy at 24 hpf), embryos are dechorionated,
stained for 15 min using 5 g/mL of acridine orange dye (Sigma), and rinsed five times

with embryo medium as described previously (Westerfield, 1993). Zebrafish embryos

m