Oil red O staining protocol

1. More than 200 worms are collected to perform the fat staining. Wash worms from seeded plates with WWB (1ml 1 x Sbuffer + 0.001% Triton), and centrifuge the mixture at 4000rpm for 30s to remove the Then wash worms for two times with 1 ml 1 x Sbuffer, and centrifuge the mixture at 4000rpm for 30s to remove the supernatant. Remain 90ul mixture in the tube.

2. Add 10ul paraformaldehyde stock solution (10%) to the 90ul mixture, and froze the samples at -80℃ for 10 min (longer if necessary). Warn: the paraformaldehyde need to be preheated at 55℃ for 10 min.

3. The samples are frozen in dry ice/ ethanol and thawed under a stream of warm water for three cycles. Remove all the supernatant as much as possible. Wash worms with 100ul 1 x Sbuffer for two times, and centrifuge the mixture at 4000rpm for 30 second, then remove all the supernatant as much as possible.

4. Add 500ul working solution of Oil red O, and staining for 30 min at room temperature, gently suspend worms every ten minutes. Then centrifuge the mixture at 4000rpm for 1 min to remove all the supernatant as much as possible.

5. Add 100-200ul 1 x Sbuffer to wash worms more than 2 hours on ice, then use 10ul mixture to take photos. Warning: Cannot use the centrifuge to settle the worms.

Stock solution (5mg/ml): 100mg Oil red O + 20ml isopropanol

Working solution: 6ml stock solution + 4ml ddH₂0