Development of a Hypoxia-Induced Zebrafish Choroidal Neovascularization Model - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 201

instructions. Whole mount immunostained zebrafish were sectioned sagitally (5 m

thick). Slides were then counterstained by nuclear fast red (Sigma, St. Louis, MO).
Since JB-4 ordinarily inhibits penetration of large molecules, such as antibodies,
instead of conventional immunohistochemistry, we stained and then sectioned
zebrafish. Sections were examined using a Zeiss Axiostar compound microscope
(Zeiss).

m

16.2.7 RT-PCR

To determine if HIF-1 was upregulated after CoCl 2 treatment, total RNAwas isolated

from control and treated zebrafish and reverse transcribed with MMLV reverse
transcriptase (GIBCO/Invitrogen, Carlsbad, CA) primed with oligo dT and subjected
to PCR using zebrafish-specific primers. Primers included HIF (left: 5 0 -GAC GTG
GAAGGT TCT TCACTG-3 0 ;right:5 0 -TCA AGAGGT CAT CTG GCT CAT-3 0 )and
VGEF (left: 5 0 -GTA AAG GCT GCC CAC ATACC-3 0 ;right:5 0 -GCT TTG ACT TCT
GCC TTT GG-3 0 ).

a

-actin was used as an internal control. Semiquantitative PCR
was performed using Advantage 2 Taq Polymerase (BD Biosciences, Palo Alto, CA)
in a PTC-100 thermocycler (Bio-Rad, Hercules, CA) using the following cycling
parameters: 94 C for 2min followed by 30 cycles of 94 C/1min, 59 C/30 s, and
72 C/1min, followed by 72 C for 10min. PCR products were visualized following
electrophoresis in a 2.0% agarose gel and stained with ethidium bromide.

b

16.2.8 Whole Mount In Situ Hybridization

To determine the site of HIF-1 a upregulation, using the manufacturer's protocol

(Roche Diagnostics, Inc., Indianapolis, IN) and HIF-1 a

cDNA as template, a RNA
probewas synthesized and digoxigenin-labeled for wholemount in situ hybridization.
Zebrafish were fixed with 4% paraformaldehyde in PBS and rehydrated with PBST.
RNA probe was hybridized at 65 C in hybridization solution (50% formamide,
5 SSC, 0.1% Tween 20, 0.05mg/mL heparin, 0.5mg/mL tRNA, 10mM sodium

citrate buffer, pH 6.0). Alkaline phosphatase-conjugated anti-digoxigenin antibody
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used for detection. For
staining, zebrafish were equilibrated in NTMT buffer (0.1M Tris-HCl, pH 9.5,
50 mM MgCl, 0.1M NaCl, 0.1% Tween 20) at room temperature. Then, 4.5

m Lof

75 mg/mL NBT (nitro blue tetrazolium) and 3.5 L of 50mg/mL BCIP (5-bromo-4-

chloro-3-indolyl phosphate) per mL were added to the staining solution. The staining
reaction was stopped by washing zebrafish with PBST. Zebrafish were then examined
on a Zeiss Stemi 2000C stereomicroscope (Zeiss).

m

16.2.9 Drug Treatment

Celebrex, genistein, and SU5416 (Calbiochem/EMD, Gibbstown, NJ) are known to
cause antiangiogenic effects in mice (Fong et al., 1999; Leahy et al., 2002). We next