BODIPY 576/589

Protocol for BODIPY 576/589 staining with microscopy ^[1]
1. Autoclave coverslips in a glass bottle.
2. In the tissue culture hood, place coverslips into 35 mm cell culture dishes.
3. Prepare 2 mg/ml collagen solution in PBS.
4. Treat the coverslips with collagen to promote cell adherence. Add 3 ml collagen solution to culture dishes and incubate at 37 °C for 30 min.
Note: Use forceps to ensure that coverslips are flush with the bottom of the culture dish, eliminating any air bubbles that may be under the cover slips.
5. Aspirate the collagen solution.
6. Wash with PBS.
7. Add PBS to culture dishes and place under UV light in the culture hood to sterilize.
8. Plate cells into culture dishes containing the coverslips. The optimal cell number should be determined to achieve confluence of 30-50% at the time of staining to permit proper imaging. For A498 cells used in this protocol, 100,000 cells were plated in 35 mm wells to permit staining at 48 h post plating.
9. Incubate under the culture conditions relevant to your experiment.
   a. For this protocol, A498 cells were incubated in DMEM (high glucose, L-glutamine, sodium pyruvate) supplemented with 10% FBS at 37 °C.
   b. Overnight incubation of cells with 30 μM oleic acid with BSA can serve as a positive control for increased neutral lipid content, as oleic acid is a potent inducer of triglyceride synthesis and storage. Fatty acid free BSA serves as a control.
10. At the time-point of interest, prepare 2 μM BODIPY 576/589 staining solution in PBS.
   a. For this protocol, A498 cells were stained 48 h after plating, after an overnight incubation with BSA or BSA + oleic acid.
11. Wash cells with 3 ml PBS.
12. Incubate on 3 ml staining solution for 15 min at 37 °C.
Note: From this point, protect samples from light as much as possible.
13. Wash twice in 3 ml PBS.
14. Fix cells in 3 ml 4% PFA for 30 min at room temperature.
15. Remove 4% PFA.
16. Wash samples 3 x 5 min in PBS.
17. Use forceps to mount cover slips onto glass slides.
Use forceps to pick up cover slips and place onto the drop of mounting solution, ensuring that the side that side with cells is placed face down onto the glass slides.
18. Allow the mounting solution to cure overnight at room temperature.
19. Immediately image cells.
BODIPY 576/589 photobleaches rapidly. Including 200 ng/ml of BODIPY 576/589 in the medium during imaging can minimize this problem. Additionally, using the stable Hoechst staining in the blue channel to find, focus, and center fields before imaging BODIPY 576/589 ^[2].
2 μM BODIPY staining solution：
a. Prepare 5 mM BODIPY stock solution
Dissolve 1.3 mg BODIPY in 1 ml DMSO and can be stored at -20 °C, protect from light.
b. 2 μM BODIPY staining solution can be prepared by diluting stock solution 1:2,500 in PBS.

This protocol only provides a guideline, and should be modified according to your specific needs.