Paraformaldehyde for Drosophila (4%)

MATERIALS

NaOH (one pellet; ~0.2 g)

Paraformaldehyde powder (8 g)

PB (0.1 M phosphate buffer pH 7.2)

Sodium phosphate dibasic stock (0.5 M)

Sodium phosphate monobasic stock (0.5 M)

Beakers (100-mL, 250-mL)

Fume hood

Hot plate

pH paper

Stir rod

1. Place the paraformaldehyde powder and NaOH pellet in a 250-mL beaker.
2. Prepare 100 mL of 0.1 M sodium phosphate dibasic: Use 20 mL of 0.5 M sodium phosphate dibasic stock and bring to 100 mL with H₂O. Add this to the beaker from Step 1.
3. Stir while heating on a hot plate under the fume hood until all the paraformaldehyde powder is dissolved. Do not boil. Turn off the heat.
4. Prepare 100 mL of 0.1 M sodium phosphate monobasic: Use 20 mL of 0.5 M sodium phosphate monobasic stock and bring to 100 mL with H₂O.
5. Slowly add 0.1 M sodium phosphate monobasic from Step 4 to the paraformaldehyde solution from Step 3 until the pH is 7.2 (check with pH paper).
6. Measure the volume of 0.1 M sodium phosphate monobasic left unused. Add this volume of PB to the fixative to give a total fixative volume of 200 mL.
7. Store at 4°C and discard after 5 d.