Salivary Gland Dissection/ Fixation

1. Place the larvae in 1x PBS( in a small dish or cover slip) 2. Grasp the mouth hooks firmly with one pair of forceps 3. Then grasp the body of the larva approximately midway down with the other pair of forceps 4. Slowly pull the two pairs of forceps apart 5. The mouth hooks should separate from the body, with the salivary gland 6. The paired salivary glands are large, clear sausage like shaped cylinders connected at one end by a common duct. 7. Try to keep the glands attached to each other by the duct 8. Remove the mouth hooks and any other unwanted tissue by gently teasing with the forceps 9. (Optional step for making polytene squish: Using a transfer pipet, put several drops of 45% acetic acid into a clean plate and then transfer a larva into the acetic acid.) 10. Place salivary glands in new dish of PBS. Remove the fat bodies (take care not to damage the glands). 11. Once the fat bodies are off, place the glands in a new dish of PBS. 12. Transfer the dissected salivary glands into an Eppendorf tube, with 4% formaldehyde in PBS 13. Rock for 20 minutes 14. Wash 3x with 1 ml 1xPBS for 5 minutes. 15. Rinse 2x with 1 ml methanol 16. Place 1 ml methanol in tube and place at -20oC till used for in situ. (Be sure the tube is labeled with date, contents and initials on both the top and the side of the tube).