Detection of nitric oxide by diaminofluorescein visualizes the skeleton in living zebrafish.

Several in vivo stainings, such as Calcein, Alizarin Red and
Quercetin are commonly used to visualize ossification in
living teleost specimen. These staining techniques represent
important tools for bone research in fish, but do not visualize
cartilage. In the present study, we show that nitric
oxide (NO) labelling by DAF-FM DA visualizes both bone
and cartilage in vivo during zebrafish skeletogenesis. NO
detection performed in Tg(osterix:mCherry) or in combination
with Alizarin Red in wild-type zebrafish reveals that
intense staining through NO labelling colocalizes with the
appearance of osteoblasts and characterizes ossified structures.
Cartilage structures are clearly distinguished in the
living larvae, although the labelling is less intensive when
compared to ossified structures. This method is the first
and easy to handle alternative to cartilage and bone double
stainings on fixed samples. In contrast to most live skeletal
stainings, which only stain the mineralized bone structures,
this protocol in addition allows in vivo visualization of
cartilage.