Development of a Hypoxia-Induced Zebrafish Choroidal Neovascularization Model - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 200

(Seng et al., 2004), was generated by Phylonix (Cambridge, MA). Rhodamine-
conjugated secondary antibody was purchased from Jackson ImmunoResearch
Laboratories, Inc. (West Grove, PA). Horseradish peroxidase (HRP) suppressor,
HRP-conjugated secondary antibody, and Ps-atto were purchased from Beckman
Coulter/Lumigen, Inc. (Southfield, MI).

16.2.3 CoCl 2 Treatment to Induce Hypoxia

CoCl 2 (Sigma, St. Louis, MO) was dissolved in fish water and serially diluted to the
desired concentration and used to treat 24hpf zebrafish continuously for 4 days. On
day 5, treated zebrafish were processed to perform the following procedures.

16.2.4 Whole Mount Immunofluorescence Staining
and Fluorescence Microscopy

To visualize vessel pattern in zebrafish eyes, whole mount immunofluorescence
staining was performed. Zebrafish were fixed by Dent's fixative (DMSO/methanol ¼

1/4) and processed for whole mount immunochemical staining following standard
procedures (Westerfield, 1993). Phy-V antibody was used as the primary antibody.
Rhodamine-conjugated secondary antibody was then used to stain zebrafish. Animals
were examined using a Zeiss M2Bio fluorescence microscope (Zeiss, Thornwood,
NY) equipped with a red rhodamine cube, and a chilled CCD camera (AxioCam
MRm, Zeiss). Images were analyzed with AxioVision software Rel 4.0 (Zeiss) and
Photoshop 7.0 (Adobe, San Jose, CA).

16.2.5 Eye Extraction

Whole mount Phy-V-stained zebrafish were incubated with collagenase (150 unit/
mL) at 37 C for 40 min in an Eppendorf tube to loosen eyes from sockets without
damaging vessels. An Eppendorf tube containing treated zebrafish was then gently
shaken a few times and loosened, intact eyes were then dissociated from whole
zebrafish. The CVP, located in the back of the eyes, was then accessible for assessing
drug effects.

16.2.6 Immunohistochemistry and Microscopy

Immunohistochemistry was used to validate the CNV model and to assess drug
effects. Briefly, whole mount immunostaining was performed as described above;
however, instead of rhodamine-conjugated secondary antibody, HRP-conjugated
secondary antibody and DAB (both from Jackson ImmunoResearch Laboratories,
Inc.) were used to stain zebrafish. After staining, zebrafish were embedded in JB-4
(Polysciences, Warrington, PA), a transparent medium, following manufacturer's