Hematopoietic and Vascular System Toxicity - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 80

23. Prestain for 5min in staining buffer (100mM Tris (pH 9.5), 50 mMMgCl 2 ,
100mM NaCl, 0.5% Tween 20 in water).
24. Make staining mix (225 m L of 50 mg/mL nitro blue tetrazolium chloride

L of 50mg/mL 5-bromo-4-chloro-3-indolyl phosphate,
toluidine salt (BCIP) in 50 mL staining buffer).
25. Stain until color develops in relevant blood and vascular tissues.
26. Wash off with PBT.
27. Maintain in 4% PFA.

(NBT) and 175 m

Protein levels are difficult to assay as antibodies against zebrafish proteins are not well
developed. If there is an antibody that recognizes a zebrafish protein of interest,
immunohistochemical analysis can be performed on whole mount embryos or adult
kidney sections (Vasilyev et al., 2009). An alternative approach to looking at protein
level involves transgenic models. Many transgenics have been created where a
fluorophore has been attached to a protein of interest or, more commonly, is expressed
under the control of a promoter for a gene of interest. These fluorophores include, but
are not limited to, GFP, dsRED, andmCherry. Fluorophore levels can be assayed using
fluorescence or confocal microscopy. Alternatively, levels can also be measured using
antibodies or RNA probes that recognize the fluorophore. Beyond these methods,
there are alternative experimental approaches specific to one or more blood cell types.
These are described below.

7.3.5.2 Hematopoietic Stem Cells Definitive HSCs express c-myb, runx1,
and low levels of CD41, so HSC number and location can be determined by runx1 and
c-myb ISH. Probes for runx1 and c-myb RNA demonstrate that HSCs first appear at
36hpf in the AGM, and exist throughout zebrafish development and life. Since c-myb
also stains primitive blood cells in the ICM, adjacent to the AGM, staging embryos at
36-40hpf is important for this assay.

To assay protein level, some transgenic lines have been developedwith promoters
of these genes. ACD41-gfp transgenic (Lin et al., 2005) can be used to visualize HSCs
in the AGM and CHTof the developing embryo (Kissa et al., 2008). Myb-GFP (North
et al., 2007) and runx1-EGFP (Lam et al., 2009) transgenics have also been recently
created.

7.3.5.3 Erythrocytes Embyronic erythrocytes, from both primitive and
definitive hematopoiesis, express gata1 and embryonic globins e1.

These RNAs can be visualized from five somites (de Jong and Zon, 2005). In the
adult zebrafish, erythrocytes express gata1 and adult globins

a e3 and b

a1. Gata1
transgenic lines, where GFP (Long et al., 1997) or dsRED (Vogeli et al., 2006) is under
control of the gata1 promoter, can be used to visualize erythrocytes. In addition to
these tools, o -dianisidine staining can be used for looking at hemoglobin levels.
Presence of hemoglobin enhances o -dianisidine oxidation by hydrogen peroxide
(Paffett-Lugassy and Zon, 2005).

a a1 and b