Home Page        FAQs       Streisinger        Zebrafish ABZs        Lab Protocols        Stages        Methods        Resources        Contacts

Part 2.  Specific Staining of Larval Cartilages

(This is at least a 43 minute procedure, so don't put off getting started until near the end of the lab!)

This procedure requires that we euthanize the larva and you should consider the ethics of such use of a vertebrate animal (see Ethical Concerns). If you believe, as we do, that the learning experience justifies this use, then carry out the first steps with care to see to it that your specimen is completely anesthetized before you add the fixative. It is highly likely that at this early larval stage of a developing teleost, no pain sensation exists at all. Even if this assertion is incorrect, if you follow the protocol properly, there will be no issue of discomfort or pain.

If you believe the euthanasia not justifiable, you may not want to carry out this part of the exercise. There will be no attendant penalty. Simply turn in a write-up explaining your viewpoint in place of the drawing requested below.

Staining Protocol

Place one or more deeply anesthetized larva(e) in a small droplet of dilute anesthetic in a capped tube. Carry ou the following steps at room temperature. Times given are minimal: longer will do no harm and might improve the quality of the preparation. During each time interval, cap the tube and mix occasionally by inverting.

• Euthanize and fix for 10 minutes or more by adding an excess of 5% tritchloroacetic acid
• Wash for 3 minutes with Acid-Alcohol (0.37% HCI, 70% ethanol)
• Stain 10 minutes with 0.1% Alcian Green. If you have time, let the reaction go longer.
• "Differentiate" with Acid-Alcohol (that is, wash the stain out of noncartilaginous tissues) for at least 10 minutes.
• Clear in Glycerol-KOH (50% glycerol, 0.25% KOH) for 10 minutes. You will see the tissues becoming transparent.
• Inspect the larva, placed in a petri dish and immersed in Glycerol-KOH. Use the dissecting microscope. If you like, you could also then make a bridged mount on a slide in the same solution, and inspect your preparation with the compound microscope.
• Make a drawing of the pattern of cartilages that you see and identify their locations (that is, show in exactly what parts of the organism the cartilages are positioned).
• Deduce what supportive or protective (or other) function(s) you suspect particular elements may have and note this clearly on your drawing. The instructors will be please to comment on your accuracy if you desire, and tell you the names of the individual elements.

For your lab report for Part 2, submit your annotated drawing and accompanying notes and comments.

Go to Part 1
Go to Laboratory Protocol page