780 confocal microscope

Cells were cultured on slices in 6-well plates and fixed with 4% paraformaldehyde for 10 min at room temperature. Then, the cells on slice were rinsed with PBS and permeabilized with 0.2% Triton X-100 PBS solution for 5 min, followed by blockade with 5% BSA for 1 h. Cells were rinsed once with PBS and were incubated with primary antibodies according to the manufacturer's instruction. Then, cells were rinsed with PBS three times, and incubated with secondary antibody (Alexa-Fluor 488-conjugated and 555-conjugated antibodies) for 1 h. For nuclear staining, 4′,6-diamidino-2-phenylindole (DAPI) was incubated at 37°C for 10 min. Zeiss 780 confocal microscope was used to obtain immunofluorescence images processed with Adobe Photoshop for publishment.

3T3-L1 adipocytes expressing control shRNA or shRNA (#1) targeting Tip60 were serum-starved overnight and treated with BSA or 0.25 mM OA in high glucose serum-free DMEM for 2 h. Cells were then fixed with 4% paraformaldehyde, rinsed with PBS for two times, permeabilized with 0.1% Triton X-100 (diluted in PBS) for 10 min at 4 °C and then blocked with 5% BSA for 30 min. Cells were rinsed two times with PBS and were incubated with primary antibodies to lipin 1 (Santa, B-12) and Calnexin (Proteintech, Cat. 10427-2-AP) overnight at 4 °C. The cells were then rinsed with PBS three times, and incubated with Alexa-Fluor 488-conjugated and 594-conjugated secondary antibodies for 6 h. Fluorescence images were obtained with a Zeiss 780 confocal microscope and processed with Adobe Photoshop for presentation.