To observe intracellular expression of p-Nrf2, cells cultured on glass coverslips were pretreated with or without FST for 1 h and additionally exposed to H2O2 for 24 h. After fixation and permeabilization, the cells were incubated anti-rabbit p-Nrf2 antibody (Abcam, Inc., Cambridge, UK), and then proved with Alexa Fluor 647-conjugated secondary antibody (Abcam, Inc.). Additionally, nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI; Sigma-Aldrich Chemical Co.). The stained cells were observed under a fluorescence microscope as previously described [35 (link)].
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