Las software

Cells were labelled with the plasma membrane-incorporating dye di-4-ANNEPDHQ (Thermo Fischer Scientific, D36802) at the concentration of 1 μM for 10 min. Image acquisition was carried out in a TCS SP5 laser scanning confocal microscope with Leica LAS software (Leica), using a 63× oil-immersion objective. An argon-ion laser at 488 nm was used for excitation, and the detection ranges of PMTs were set to 500–580 nm and 620–720 nm, respectively for the two emission channels. The GP values were calculated according to the following equation:
GP = (I _ (500 − 580) − G I _ (620 − 750))/(I _ (500 − 580) + G I _ (620 − 750)).
Where I represents the intensity of each pixel in the spectral channel (in nm) and G represents the calibration factor, which compensates the differences in the efficiency of collection in the two channels. In order to select the signal originating only from the plasma membrane we segmented our images with an image analysis software (CellProfiler, http://cellprofiler.org). The end product of this procedure was a plasma membrane segment which was originating from one cell. Each individual experimental group contained at least 80 membrane segments (80 cells) for one experiment. The measurements were repeated in three independent experiments.

SkMel2 cells stably expressing ezrin-GFP were detached using Versene/EDTA, resuspended and maintained in DMEM for 1 h, seeded onto imaging slides (µ-Slide 8-well, glass bottom, Ibidi) coated with 2% BSA, fixed with 4% formaldehyde and carefully washed twice with PBS. Interference reflection imaging^{48 (link)} was performed on a Leica TCS SP8 confocal microscope. Reflection and fluorescence images were obtained using 488 nm excitation and 480–497 nm or 503–559 nm emission wavelengths, respectively. To produce an IRM image, the focus was adjusted near the glass surface to obtain a zero-order interference image and the detector was adjusted for best contrast. For the reflection image, the pinhole was opened to 4 airy units (AU). In addition to the reflection image, a stack of 25 confocal fluorescence images was recorded in the GFP channel with the pinhole set to 1 AU. Top and side views of the stacks were reconstructed using the Leica LAS software and further processed using ImageJ⁴⁶ .

The blend mixtures after dialysis procedure were observed and optical images were collected from the Leica EC3 stereo microscope equipped with Leica LAS software (Leica, Wetzlar, Germany). The mixtures in their containers were placed on a stage with a dark base and the pictures of the blend solutions were captured using the digital camera zoomed onto the solutions as closely as possible.

LipidGreen staining was performed at 15 dpf to check for the presence of adipocytes. Larvae were fasted for 12 h before staining in order to empty the digestive tract, as the presence of food in the gut may give a high background signal, thereby making it difficult to visualize adipocytes. The protocol for LipidGreen staining was performed as described in Chun et al., 2013 [55 (link)]. LipidGreen solution with a final concentration of 1 µM was prepared in E3 medium. After staining, larvae were 3× 10 min washed with E3 medium. During staining and washes, larvae were kept protected from light. Larvae were anaesthetized with 0.15% MS222 and mounted on 3% methyl cellulose containing 0.15% MS222. Images of larvae were captured using an inverted fluorescence microscope (Leica DMIL LED with a DFC295 camera, Leica Microsystems BV, Rijswijk, The Netherlands) and Leica LAS software.

Hemoglobin staining was performed as described previously^{27 (link)}. Dechorionated live embryos at 72 hpf were stained in 0.6 mg/mL o-dianisidine (Sigma-Aldrich, St. Louis, MO) containing 0.01 mol/L sodium acetate (pH 4.5), 0.65% H₂O₂, and 40% ethanol in the dark for 30 min. Embryos were then dehydrated through graded ethanol washes of 50%, 75%, and 100%, for 5 min each. Finally, embryos were cleared in benzyl benzoate and benzyl alcohol at a 2:1 ratio. Embryos were stored in the clearing solution until imaged using a Leica DFC340FX fluorescent microscope with PlanAPO 1.6×/0.05 NA objective (Leica Camera Inc., Allendale, NJ, USA). Image capture was done with Leica LAS software, and post processing was done using Adobe Photoshop CS4 (Mountain View, CA, USA).