Nile red

The larvae were washed and subsequently stained with Nile Red (Sigma, Jerusalem, Israel), which specifically stained neutral lipids, such as triglycerides [64 (link)]. The larvae were exposed to Nile Red (500 ng/mL) for 0.5 h and subsequently euthanized. Using a 1% low melting point agarose or a glycerol-based Mounting Medium (ibidi Mounting Medium, ibidi, Grafelfing, Germany), each larva was positioned on its right side (location of adipocyte deposit) on a coverslip assembly, based on a published diagram [65 ] (wiki.zfin.org/display/prot/Viewing+Chambers, last accessed on 2 July 2023). The degree of obesity was assessed by the number of Nile Red-stained lipid droplets (assumed to be in adipocytes) and their area, as determined by confocal microscopy (Zeiss LSM 700 laser scanning confocal microscope, using an X5 objective, laser settings of 488 nm for excitation and ≥539 nm for emission and ZEN 2.6 (blue edition) software (Zeiss)). Conditions, including the laser power, pinhole, master gain, and digital offset were kept constant for each experiment. The layer counted was the one with the most Nile Red-stained lipid droplets in the abdominal area. Lipid droplets smaller than 100 µm² were excluded, as were those appearing in a row (since they could be fat droplets in lymph vessels and not adipocytes).

In order to visualize SAT and VAT, fish were fasted overnight and stained with Nile red (Invitrogen, Thermo Fisher Scientific, N1142). Fish were submerged in 0.5 μg/mL (from a 1.25 mg/mL acetone stock solution) Nile red for 30 minutes and washed in clean system water for 15 minutes. To visualize SAT, adults were submerged in 10 mg/mL epinephrine in fish water for 5 minutes (45 (link)). Before imaging and dissection, fish were euthanized in ice water. Nile red–stained zebrafish larvae were examined either with a Zeiss Discovery V8 stereoscope for wide-field imaging or with a Zeiss LSM 880 confocal microscope using a 10×/0.3 NA EC Plan-Neofluar objective lens. For wide-field imaging, Nile red fluorescence was analyzed with a 470 ± 20 nm excitation filter and 525 ± 25 nm emission filter. For confocal imaging, Nile red fluorescence was excited using 514 nm laser light, and the emission was collected using a 539 to 753 nm range. Oil red O stain was used to evaluate hepatosteatosis as previously described (83 ).

For fluorescence microscopy, cells were grown at 37°C in LB medium to an OD₆₀₀ of 0.5–1.0 (unless otherwise indicated), harvested by centrifugation and resuspended in phosphate-buffered saline (50 mM; pH 7.5). The membrane was stained with Nile Red (Sigma, Darmstadt, Germany) (10 μg/ml). The cells were spotted onto a microscope slide covered with a film of 1% agarose in water. Fluorescence images were obtained with an AxioImager M2 fluorescence microscope, equipped with digital camera AxioCam MRm and AxioVision Rel 4.8 software for image processing and an EC Plan-NEOFLUAR 100×/1.3 objective (Carl Zeiss, Göttingen, Germany). The filter set 37 (BP 450/50, FT 480, BP 510/50; Carl Zeiss) and the set 43 (BP 545/25, FT 570, BP 605/70; Carl Zeiss) were applied for GFP and Nile Red detection, respectively. Images were taken with an exposure time of 1 s for the GFP constructs and 500 ms for visualization of the Nile Red stain. Pictures of B. subtilis colonies on agar plates were taken with a stereo microscope Lumar.V12 (Carl Zeiss) equipped with the ZEN lite 2011 (blue edition) software.

HeLa cells obtained from the Korean Cell Line Bank (KCLB No. 1002) were cultured in Dulbecco’s Modified Eagle’s Medium (Corning, VA, USA) supplemented with 10% fetal bovine serum (Corning, VA, USA) and 1% penicillin-streptomycin (Mediatech Inc., Corning, VA, USA). Cells were maintained at 37 °C under humified conditions with 5% CO₂ (ARA150 CO₂ incubator, Gyrozen Co., Ltd., Gimpo, Republic of Korea). For confocal microscopy, HeLa cells (1.6 × 10⁵) were seeded in microscopy chambers with coverslip bottoms (Marienfeld, Harsewinkel, Germany). After stabilization, the cells were incubated for 4 h with polymeric NPs containing PTX and Nile red (<0.1 mg/mL, Tokyo Chemical Industry, Tokyo, Japan) [37 (link),38 (link)]. During incubation, NPs were treated with 100 nM of lysotracker (LysoTracker^TM Green DND-26, Invitrogen, OR, USA) for 2 h and mounting medium was treated with 4,6-diamidino-2-phenylindole (DAPI) (Fluoroshield^TM with DAPI, Sigma, St. Louis, MO, USA). NPs were observed by confocal microscopy (Carl Zeiss-LSM800, Zeiss, Jena, Germany) (DAPI: 465 nm, LysoTracker: 509 nm, Nile red: 636 nm, 63×, ZEN2.6).

The lipophilic dye Nile red, which becomes fluorescent in lipid-rich environments [47 (link)], was obtained from Fluka (Thermo Fisher Scientific Inc., Waltham, MA, USA). It was diluted to 0.5 ng/mL in water and was added to the bacterial lawn on agar plates followed by incubation overnight in the dark at room temperature. Plates were inoculated with L4 larvae and kept for 22 h at 20 °C. Worms were then transferred to an agarose pad and immobilized in 4 µL 1% levamisole (Sigma-Aldrich, St. Louis, MO, USA). Nile red fluorescence was assessed by microscopic imaging in a Zeiss Apotome at 10x magnification (Carl Zeiss AG, Oberkochen, Germany). Fluorescence was then quantified with the help of Fiji to manually define regions of interest and determine the grey values within.

Developing seeds were collected and fixed in 2.5% glutaraldehyde and 1.6% paraformaldehyde buffered with 0.1 M phosphate buffer (pH 6.8) for 48 h at room temperature. Seeds were then dehydrated in an ethanol series, then infiltrated gradually (3:1, 1:1, and 1:3 100% ethanol: Technovit 7100, 24 h each) by using Technovit 7100 resin (Kulzer & Co., Germany), followed by three changes of pure resin. Seeds were then embedded in resin, as described by Yeung and Chan (2015 ). Sections of 3-μm thick were cut using a Reichert-Jung 2040 Autocut rotary microtome. These sections were stained with periodic acid–Schiff (PAS) procedure for structural carbohydrates and counterstained with 1% (w/v) amido black 10B in 7% acetic acid for protein (Sigma-Aldrich, St. Louis, MO, USA) or 0.05% (w/v) toluidine blue O (TBO, Sigma-Aldrich) for general histological staining (Yeung, 1984). For detecting the deposition of cuticular material in developing seeds, sections were stained with 1 μg ml⁻¹ Nile red (Sigma-Aldrich) for 1 min, then washed in running tap water for 3 min. The fluorescence pattern of Nile red was viewed under an epifluorescence microscope (Axioskop 2, Carl Zeiss AG, Germany) equipped with the Zeiss filter set 15 (546/12 nm excitation and 590 emission). All images were recorded by using a CCD camera attached to the microscope.