1. Preparation of Oil Red O Working Solution

1.1 Preparation of Oil Red O Reserve Solution. Please be advised that 500 mg of ORO powder and 100 mL of 100% isopropyl alcohol should be added to a 250 mL bottle. The contents should then be mixed thoroughly. After preparation, store the solution in a tightly sealed container away from light.

1.2 Preparation of ORO Working Solution

The ORO stock solution should be diluted with water to create a 60% isopropanol (3:2 v/v) solution.

Prior to use, the working solution should be filtered through a sterile 0.2 μm cellulose acetate syringe filter.

Please note that, for optimal solution quality, the ORO Working Solution should be prepared one day in advance and left to mix overnight. For same-day use, the diluted solution should be mixed on a shaker for a minimum of two hours. Centrifuge tubes should be wrapped in paraffin film prior to shaking in order to prevent leakage.

2. Please find below the relevant procedure for sample preparation:

2.1 The culture nematodes were prepared on Nematode Growth Medium (NGM) inoculated with E. coli OP50 (late logarithmic phase) at 20°C until the target developmental stage.

2.2. Next, add 1 mL of PBST solution (containing 0.01% Triton X-100) to the petri dish and shake gently to dislodge the nematodes from the medium. The dish should then be tilted and rinsed with 1 mL of PBST, after which the nematode suspension should be transferred to a 1.5 mL centrifuge tube.

2.3. Centrifuge at 560 × g for one minute, then discard the upper layer and keep the solid piece. The procedure should be repeated three times with 1 mL of PBST, after which the remaining fluid should be discarded and only 100 μL of the solution retained.

2.4. Next, add 600 μL of 40% isopropanol to the nematode precipitate and incubate for 3 minutes at room temperature with shaking.

2.5. Centrifuge at 560 × g for 30 seconds, discard the upper layer and reserve 100 μL only, taking care not to disturb the nematode precipitate.

Please note that for high-throughput staining, the volume of PBST should be increased. It is important to note that nematodes should not be washed in PBST for more than 15 minutes. If bacteria are still present in the serum, additional washing is required. The sample should be incubating in 40% isopropyl alcohol using a shaker or oscillator to ensure that it is well mixed.

3. The staining of liposomes.

3.1 Please add 600 μL of ORO Work Solution to each sample, then invert the centrifuge tube three times to ensure the nematodes are thoroughly mixed with the staining solution.

3.2. The product should then be incubate at room temperature for 2 hours at 30 rpm.

3.3. Centrifuge at 560 × g for one minute, discard the fluid and reserve 100 μL.

3.4. The samples should be resuspended with 600 μL of PBST and then incubated at 30 rpm for 30 minutes in order to remove excess staining solution.

3.5. Centrifuge at 560 × g for one minute, discard the fluid and reserve 50 μL.

3.6. In order to achieve optimal distinction between nematode germ cell and intestinal cell nuclei, it is recommended that 1 μL of DAPI be added per 1 mL of ORO working solution. Following a staining period of two hours, the samples should be prepared for imaging. The nuclei of the intestinal cells are larger and the gonads can be recognised by the developing germ cells.

Please note: Following ORO staining, there is a possibility that the nematodes may adhere to the inner wall of the centrifuge tube, resulting in the absence of a visible precipitate. Should this occur, please centrifuge again or leave for a minimum of 10 minutes to allow the nematodes to settle.

Please find below the fourth point on the agenda, which is entitled 'Preparation'. Please be sure to resuspend the nematode precipitate in the remaining serum, and mix thoroughly.

4 The edges of the coverslip should then be sealed with nail polish, after which the image is ready to be applied.

5. Imaging

5.1. Please use a microscope camera with colour imaging capabilities to photograph the ORO-stained nematodes.

5.2. It is recommended that a photograph be taken using a 5X objective in order to cover multiple nematode fields of view. Following this, the use of a 10X objective will allow individual nematode details to be observed.

5.3. To ensure data integrity, please export the images in TIF format.

Please note that when using ORO+DAPI staining, it is necessary to switch to fluorescence imaging mode.