X71 fluorescent microscope

The nematodes were rinsed with M9 buffer solution to remove OP50 after a 5-day treatment with 0.18 or 0 mg/mL of APPA. Afterward, 30 nematodes were treated with 100 µM of 2, 7-dichlorofluorescein diacetate (H₂DCF-DA) (Meilunbio, Shanghai, China), incubated at 35 °C for 30 min, anesthetized with levamisole, and then placed on 2% agarose [52 (link)]. Subsequently, 30 nematodes per group were measured and detected by blue fluorescence (Ex 430–455 nm) under the Olympus X71 fluorescent microscope (Tokyo, Japan), viewed through a ten-fold objective. The nematode size was determined from the bright field image. Finally, their relative fluorescence intensity was determined by the integrated density and quantitative fluorescence intensity by ImageJ 15.2v [63 (link)]. Then, the fluorescence intensity of nematodes per unit area was calculated. The data were analyzed in terms of the fluorescence intensity of the experimental group relative to the blank group.

On day 12, following treatment with or without 0.18 mg/mL APPA, the nematodes were narcotized using 10 mM levamisole (Aladdin, Shanghai, China). Subsequently, 30 nematodes per group were measured and detected by purple fluorescence with an excitation wavelength of 400–430 nm under the Olympus X71 fluorescent microscope (Tokyo, Japan), viewed through a ten-fold objective. The nematode size was determined from the bright field image. In addition, the relative fluorescence intensity of the nematodes in vivo was measured using the Image J 15.2v program, then the fluorescence intensity of nematodes per unit area was calculated. The data were analyzed in terms of the fluorescence intensity of the experimental group relative to the blank group.

The oil red O stain solution was prepared by combining 2% Triton X-100 with 1% oil Red O solution (Aladdin, Shanghai, China). Next, 30 nematodes were treated with 0.18 or 0 mg/mL of APPA for five days, then rinsed in two changes of M9 buffer, fixed for 20 min with the oil red O stain solution, washed again with M9 buffer, and placed on a 2% agarose gel mat. Subsequently, 30 nematodes per group were measured and detected under the Olympus X71 fluorescent microscope (Tokyo, Japan), viewed through a ten-fold objective. In addition, the mean oil red O intensity in nematodes was measured using Image J 15.2v [63 (link)].