Use of Stains in Screening

Structural stains: A number of stains have been described in vertebrate systems that bind particular cellular or extracellular components. These types of stains can be used to reveal particular aspects of fish anatomy that may not be visible on inspection, such as the vertebral column and head skeleton (17). A number of fluorescent stains are available that stain nuclear or cell membrane components that are detectable by fluorescent microscopy. These are included for reference, but clearly may not be accessible techniques for every lab and may not be suitable for use in a larger screen.

3.3.3.1. Alcian Blue

This stain binds to a matrix component of cartilage that begins to develop during the hatching period. Staining in embryos and early larvae reveals the pattern of the developing fin cartilage and head skeleton. Subtle changes in the patterning of these structures can be revealed by using simple light microscopy without the need for dissection.

1. Fix early larvae (3-5 d) in 10% buffered formalin (pH 7.0) for 3 h to overnight.

3. Place in 0.1% (w/v) alcian blue overnight at room temperature.

4. Rinse in EtOH and rehydrate with PBS.

5. To clear tissue, place in dilute trypsin solution (~0.05% w/v) and leave at room temperature until embryos become soft and transparent. The eyes can then be removed, and the embryos mounted in glycerol. (This step is optional and required only when looking in detail at cartilage.)

6. Pigmentation may be bleached by placing stained specimens in 0.35% H2O2 dissolved in 0.1 M KOH.

3.3.3.2. Alizarin Red

This stain binds to forming bone (17). Although most bone develops relatively late, researchers may wish to analyze the affects of mutation on early ossifications.

2. Stain in 0.001% (w/v) alizarin red/1% (w/v) KOH for 3 h.

3. Rinse and store in glycerol.

3.3.3.3. Acridine Orange

This stain can be used to access the amount of cell death in a given mutation.

1. Soak live embryos in ~0.1 mg/mL solution for 2-3 min.

2. Examine immediately with fluorescein filter set.

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