Tcs sp5 fluorescent microscopy

The MDA-MB-231 cells were plated in 24-well plates with a round coverslip in each well, the plates were stored overnight at 37 °C in order to cells adhere in the round coverslip. After the cells were treated with DHA, Delta-T3 or their co-treatments for 24 h, cells were washed once with PBS and fixed with paraformaldehyde (4%) for 10 min. Next, they were washed three more times with PBS. Next, the cells were incubated with Bodipy dissolved in PBS at 1:300 (v/v), for 30 min at room temperature in the dark. The cells were washed 3 times with PBS. Then cells were washed three times with PBS, and 300 μL of 4 ‘solution, 6-diamidino-2-phenylindole (DAPI) at 1:5000 in PBS (v/v) was added and the cells were incubated with this solution for 5 min. Then they were washed 3 times with PBS. The round coverslips with the cells were fixed on the microscope slide with prolong according to the manufacturer’s (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA) instructions. The images of the LD were obtained by confocal microscopy (Leica TCS SP5 fluorescent microscopy). The capture of images by Leica TCS SP5 fluorescent microscopy was made with an increase of 63× and zoom of 4.