Hematopoietic and Vascular System Toxicity - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 78

7.3.4 Confocal Microangiography

Brant Weinstein and colleagues developed microangiography for assaying flow in the
vasculature of zebrafish embryos (Weinstein et al., 1995). By visualizing blood flow in
the embryo, any vasculature defect can be identified and localized. This technology
has also been developed using quantum dots (Rieger et al., 2005), but here we will
discuss microangiography using fluorescent beads. This approach was used to
develop the “Interactive Atlas of Zebrafish Vascular Anatomy” (Isogai et al., 2001),
and a more detailed methodology can be found at this site.

Confocal Microangiography

1. Fluorescein-tagged carboxylated latex beads, 0.01 m in diameter, are diluted

1:1 in a 2% BSA solution, sonicated, and spun for 2min at full speed.
2. One- to seven-day-old zebrafish embryos/larvae are anesthetized with tricaine.
3. Beads are injected into the sinus venosus (in a 1-3dpf embryo) or directly into
the cardiac ventricle (4-7dpf embryo).
4. Beads will spread throughout the entire fish within minutes, but will begin to
cluster. Subsequently, confocal imaging must be performed immediately.
Embryos are mounted in methylcellulose or agarose. Images of optical
sections are collected with 2-5

m

m m between sections.

7.3.5 RNA and Protein Level

Another approach to toxicity testing in the hematopoietic and endothelial systems is
by determining RNA and protein levels of key markers in these cells. In this section,
we will describe methods used to analyze RNA and protein levels in the zebrafish. For
each blood and endothelial cell type, we will describe specific genes and tools that are
used to identify the cell of interest. For toxicity testing, these assays can be used to
ensure that blood and vascular development is not delayed or blocked after treatment
with compound.

7.3.5.1 Assays RNA level is commonly assessed in zebrafish embryos by
whole mount in situ hybridization (ISH). ISH involves first developing an
antisense dig-labeled riboprobe against your RNA of interest. Templates for this
process can often be clones of your gene of interest, readily available for all discussed
RNAs at zfin.org. The probe generated is then hybridized in thewhole mount embryo,
and staining localizes RNA expression to specific cell types. A similar approach has
also been used on adult zebrafish tissue sections (Smith et al., 2008). The protocol
below, describing probe preparation and whole mount in situ hybridization of the
embryo, is described in more detail in Thisse and Thisse (2008).

Making an Antisense Probe (Paffett-Lugassy and Zon, 2005)

1. Digest template plasmid to linearize DNA. Confirmcomplete digest by running
on an agarose gel. Alternatively, generate a PCR product of your template.