Slowfade diamond antifade mounting media

Cells were grown on glass coverslips, treated as required and fixed with 4% paraformaldehyde. Lipid droplets were visualized by staining with 0.1 μg/mL Nile Red (Santa Cruz) or HCS LipidTox green (1:200, Invitrogen H34475). For co-staining, cells were fixed with 4% paraformaldehyde, blocked for 1h in IF solution (PBS, 2% BSA, 0.1% Triton-X100), and incubated overnight with mouse a-myc (1:250, Cell Signaling 2276S), followed by donkey anti-mouse Alexa Fluor 594 secondary (1:400, Invitrogen R37115), donkey anti-rabbit Alexa Fluor 488 (1:400, Invitrogen A21206) and LipidTox green (1:200, Invitrogen H34475) for 1 hour at room temperature. Finally, cells were incubated with Hoechst 33342 nuclear stain for 5 minutes, and mounted with SlowFade Diamond antifade mounting media (Life Technology S36968). Slides were imaged on an Olympus FV3000 confocal microscope or a Zeiss Axioskope wide field microscope. Lipid droplet numbers and sizes were quantified with ImageJ and the particle counting plugin.

All animal work was approved by OSU’s Institutional Animal Care and Use Committee (IACUC). Five million cells in PBS were injected subcutaneously on the back of 7–9 week old female nude mice (Charles River) and tumor growth was measured weekly using calipers.
Excised tumors were snap frozen in OCT medium and cryosections were prepared at the OSUCCC Comparative Pathology and Mouse Phenotypic core. For immunohistochemical detection of Ki67 and cleaved Caspase 3 slides were equilibrated at room temperature for 20 minutes, fixed with 4% paraformaldehyde and blocked for 1 hour in IF solution (PBS, 2% BSA, 0.1% Triton-X100). Slides were then incubated overnight with rabbit anti-ki67 (1:250, Abcam ab15580) or rabbit anti-cleaved caspase 3 (1:250, Cell Signaling 9664S), followed by donkey anti-rabbit 488 (1:400, Life Technologies A21206) or goat anti-rabbit 594 (1:400, Life Technologies A11012), respectively, for 1 hour at room temperature. Finally, nuclei stained with Hoechst 33342 and mounted with Slowfade Diamond antifade mounting media (Life Technology S36968). Slides were imaged with a Zeiss Axioskop microscope equipped with a CCD camera.