Salivary Gland Preparation (Squash Technique)

• Fruit fly larva (wild-type and tandem-duplication mutants)
• Ringer’s insect saline
• Fine forceps and probe
• Aceto-orcein
• Dissecting and regular microscopes
• Slides, coverslips
• Small dish of melted paraffin
• Paintbrush

1. Select a third instar larva, for which the cuticle has not yet hardened, from a wild-type culture of Drosophila. Place it into a drop of Ringer’s saline solution on a slide.
2. Place the slide on the stage of a dissecting microscope and view the larva with low power. Grasp the anterior of the larva with a fine-point forceps and pin down the posterior portion with a probe. Gently pull the head off and discard the tail of the larva.
3. Locate the salivary glands and their attached fat bodies. The glands are semitransparent and attached by ducts to the digestive system. The fat bodies are white and opaque. Tease away the fat bodies and discard.
4. Place a drop of aceto-orcein on the slide next to the Ringer’s and move the salivary glands into the stain. Blot away any excess Ringer’s.
5. Place a coverslip over the preparation and allow it to stand for 1–3 minutes (it will take a few trials to obtain properly stained chromosomes). Gently squash the gland preparation in the following manner:
   • Place the slide between several layers of paper toweling.
   • Place your thumb on the top of the towel immediately over the coverslip and gently roll your thumb while exerting a small amount of pressure (as though you were making a fingerprint). Do not move your thumb back and forth. One gentle roll is sufficient.
   • Remove the slide from the towels, and seal the edges of the coverslip by using a paintbrush dipped in melted paraffin.
6. Examine the slide with the microscope and diagram the banding patterns that are observed.
7. Repeat the squash technique using larva from a genetic variant known to be the result of a deletion and/or tandem duplication. Determine the location of the deleted or duplicated bands on the chromosomes.