Celllight er gfp

Immunofluorescence analysis was performed to examine the cellular expression of WT and mutant FBP1. FBP1-KO HepG2 cells were cultured in 4-well chamber slides (2 × 10⁴ cells/well) and then transfected with plasmids (0.5 µg). Twenty-four hours after the transfection, the cells were treated with CellLight^® ER-GFP (12 µl/well; Thermo Scientific, Massachusetts, USA), which is a marker of ER. This was followed by fixation in 100% ethanol at −20 °C for 10 min and incubation with blocking solution and primary antibodies against FBP1 (SIGMA rabbit polyclonal, clone: HPA005857), c-Myc (Santa Cruz mouse monoclonal, clone: 9E10) and GFP (MBL rabbit polyclonal, clone: 598) 48 hours after the transfection. A confocal laser microscope (LSM710, Carl Zeiss, Germany) was used to obtain fluorescence images.

Fly tissues were dissected in cold PBS and transferred to 1 µM LysoTracker Red DND-99 (ThermoFisher, no. L7528) for 3 min at room temperature. For direct imaging without co-staining of antibodies (Extended Data Fig. 7), samples were mounted and imaged within 30 min after dissection. For LysoTracker staining combined with immunohistochemistry (Fig. 1f), the samples were fixed in 4% formaldehyde for 3 min at room temperature and then stained with antibodies. LysoTracker signals in fat body tissues maintained well after co-staining with antibodies, whereas LysoTracker signals in neurons were not observed after immunostaining possibly due to the low penetration and (or) diminishment of the dye. In addition, for LysoTracker staining in the fat bodies of L3-stage larvae, LysoTracker dye could only label the acidic lysosomes that was induced under starvation condition, but not the lysosomes of the fed larvae^{32 (link),33} .
CellLight ER-GFP (ThermoFisher, no. C10590) was used to label ER in HEK293 cells. CellLight ER-GFP was dissolved in the culture medium (1:50) and incubated with cells overnight. Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 20 min at room temperature. After three washes with PBS, cells were mounted onto the microscope slides for imaging.