Immunofluorescence staining was performed as previously described [16 (link)]. The primary antibodies used were PPAR- γ (Cell Signaling Technology) and SREBP-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The secondary antibodies used were Alexa Fluor 488 and/or Alexa Fluor 594.
The SZ95 cells were fixed with 10% formalin for 15 min at room temperature. The cells were subsequently treated with 0.1% Triton X-100 in PBS for 5 min to permeabilize them. Then, the cells were blocked at room temperature for 30 min and incubated with primary antibody against SREBP-1 and PPAR-γ at 37 °C for 1 h. For double staining, a reaction with Alexa Fluor 488 phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) was performed at room temperature for 1 h. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min at 37 °C. The slides were mounted using VECTASHIELD Mounting Medium (VECTOR Laboratories, Burlingame, CA, USA).
The SZ95 cells were fixed with 10% formalin for 15 min at room temperature. The cells were subsequently treated with 0.1% Triton X-100 in PBS for 5 min to permeabilize them. Then, the cells were blocked at room temperature for 30 min and incubated with primary antibody against SREBP-1 and PPAR-γ at 37 °C for 1 h. For double staining, a reaction with Alexa Fluor 488 phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) was performed at room temperature for 1 h. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min at 37 °C. The slides were mounted using VECTASHIELD Mounting Medium (VECTOR Laboratories, Burlingame, CA, USA).
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