Zebrafish: A New In Vivo Model for Identifying P-Glycoprotein Efflux Modulators - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 172

(Bresolin et al., 2005). Furthermore, in fish, xenobiotic efflux pumps, including Pgp,
have been identified in brain microcapillaries, which are sensitive to different
glycoprotein inhibitors and substrates, and pharmacological response is similar to
mammals (Miller et al., 2005). Taken together, these data support use of zebrafish as
an animal model for assessing drug effects on Pgp inhibition.

14.2 MATERIALS AND METHODS

14.2.1 Embryo Handling

Zebrafish were generated by natural pairwise mating in our aquaculture facility as
described by Westerfield (1999). Zebrafish were maintained in embryo water (5 g of
Instant Ocean Salt with 3 g of CaSO 4 in 25 L of distilled water) at 28 C for
approximately 24 h before sorting for viability. Because early-stage zebrafish receive
nourishment from an attached yolk sac, no additional maintenance was required.

14.2.2 Reagents

Unless otherwise described, reagents were purchased from Sigma-Aldrich Co.
(St. Louis, MO).

14.2.3 Circulation Dye Injection

Zebrafish were placed on an injection stage containing fish water and immobilized by
0.32mM tricaine (ethyl 3-aminobenzoate methanesulfonate). A microinjection
needle was fabricated from a 1mm (outer diameter) glass capillary (WPI, Sarasota,
FL) using a P-30 vertical pipette puller (Sutter Instrument, Novato, CA). Using an
M-330 micromanipulator (WPI), the tip of the injection needle was inserted into the
common cardinal vein (CCV) (Isogai et al., 1994). A solution of 10mM rhodamine
123 was injected into CCVunder a stereo dissecting microscope (Stemi, 2000, Zeiss,
Thornwood, NY). Following injection, zebrafish were transferred to a Petri dish
containing fish water and incubated at 28 C for 2 h. Zebrafish were then positioned
laterally on a depression glass slide containing 1% methylcellulose for image
acquisition.

14.2.4 Fluorescence Microscopy

Fluorescence microscopy was performed using a Zeiss M2Bio fluorescence micro-
scope (Zeiss), equipped with a rhodamine cube (excitation: 540 nm, emission:
605 nm), and a chilled CCD camera (AxioCam MRm, Zeiss). Fluorescence images
were acquired at 25 magnification and processed using AxioVision software Rel 4.0

(Zeiss). ImageJ software (NIH, Bethesda, MD) was used for image analysis.