Liver Tissue Immunofluorescence Staining for Sam68

Hematoxylin and eosin (H and E) staining was performed in the liver tissue collected from Sam68^f/f and Sam68^LKO mice and fixed in 4% paraformaldehyde (PFA) by following the protocol reported by Dai et al. [51 (link)]. For assessing Sam68 expression, liver tissues were collected from HFD- or ND-fed mice, and cryosections were subjected to immunofluorescence staining. Briefly, the sections were blocked in 10% donkey serum in PBST at room temperature for 1 h, then incubated with Sam68 antibody (1:100) for overnight at 4 °C. Next day, the sections were washed with PBST, followed by incubation with Alexa Fluor 488-conjugated Donkey anti-rabbit IgG secondary antibody (Invitrogen, Waltham, MA, USA) (Green, 1:300) for 1 h at room temperature in dark. Nuclei were counterstained with DAPI (blue, Vectashield, Antifade Mounting Medium with DAPI). The fluorescence signal was examined under Nikon NI-S-E Microscope (Nikon America, Inc., Melville, NY, USA).