Dfc550 camera

Animals from same assays and age were used to implement two different staining methods. For the DAPI (4′,6-diamidino-2-phenylindole) stain, dissected gonads were fixed and permeabilized with 100% methanol at −20 °C for 5 min, and then washed in PBT (PBS + 0.1% Tween 20) three times, and stained using 100 ng/mL DAPI in PBT [18 (link)]. Germ cell nuclei were then visualized as detailed below. For the Acridine Orange stain, whole worms were stained with acridine orange in 5 mL M9 buffer supplemented with 100 µL of OP50 and 25 µL from an acridine orange stock 10 mg/mL in MQ water [19 (link)]. Worms were left in an orbital shaker at 100 rpm for one hour at 20 °C. Later, the worms were washed with M9 buffer and left in 5 mL of M9 buffer for 3 h at 20 °C to assure complete washing. Images were collected using the 10×, 20×, and 40× lenses on a Leica DM 2500 LED microscope fitted with a Leica DFC550 camera (Leica Microsystems, Wetzlar, Germany) with the Leica I3 filtercube to visualize Acridine Orange stained worms, and with the Leica A filtercube to visualize DAPI stained samples.

Quantification of lipids was performed using the Oil Red O protocol [18 (link)] with some modifications. Briefly, one day adult worms treated with different concentrations of baicalein (0, 10, 25, 50, 100 µM) were fixed in a formaldehyde PBS solution (4%) for 24 h at 4 °C. Then, the worms were washed and permeabilized using a 5% β-mercaptoethanol, 1% Triton-100 solution in tris/HCl buffer pH 7.4 for 24 h at 37 °C. Then the fixed and permeabilized worms were washed twice with PBS. Meanwhile, an ORO working solution (60%) was prepared diluting a stock solution of ORO (0.5 g ORO in 100 mL of isopropanol) in d.d. water and filtered through a 0.22 µm syringe filter. Fixed worms were treated with the ORO working solution for five minutes at room temperature, then washed three times with PBS-Triton (0.01%) buffer. ORO stained animals were mounted onto glass slides and brightfield images were taken with the 40× lens of a Leica DM 2500 LED microscope fitted with a Leica DFC550 camera (Leica Microsystems, Wetzlar, Germany). ORO quantification of the images was performed with the free software Fiji (NIH), an improved ImageJ [19 (link)]. Firstly, the images were split with the color deconvolution tool using the H AEC vector, followed by applying the thresholding tool only in the red channel and measuring the intensity in the threshold area.

The luminescent properties of OncoIr3 allowed us to track assimilation by the animals. Age synchronized L4 larvae of wild-type worms were fed with E. coli OP50 and different concentrations of the compound (0.1, 1, 10 and 100 µM) in S basal medium for 48 h at 20 ºC. Then, worms were washed with M9 buffer three times and transferred to microscope slides containing sodium azide (10 mM) as anesthetic for its visualization. Fluorescent images of the animals were taken using the A filter cube of a Leica DM 2500 LED microscope fitted with a Leica DFC550 camera (Leica Microsystems, Wetzlar, Germany) with the 10 × lens. The quantification of the compound inside the worms was performed using ImageJ software [18 (link)]. The images were split in RGB channels and only the red channel was used to measure the fluorescence of the compound inside the worms using the threshold tool of the software. Two independent assays were performed with n ≥ 10 and the statistical significance was estimated by ANOVA test.