Ottix bath

For detecting PAI-1(Plasminogen activator inhibitor-1), 4-hydroxy nonenal (4-HNE), NOX4, α-smooth muscle actin (α-SMA), Collagen Type I Alpha 1 Chain (COL1A1), and p-SMAD2/3 by fluorescence immunohistochemistry assay, formalin-fixed and paraffin-embedded sections of mouse tissues were dewaxed in an OTTIX bath (Diapath) and were blocked with solution containing 5% BSA (GenDEPOT) and 0.1% Triton 100× (Merky). Slides were incubated with a primary antibody mixture of anti-PAI-1 rabbit IgG (Santa Cruz Biotechnology), anti-4-HNE rabbit IgG (Abcam), and anti-NOX4 rabbit IgG (Santa Cruz Biotechnology) or with a mixture of anti-α-SMA mouse IgG (Sigma Aldrich), anti-COL1A1 mouse IgG (Santa Cruz Biotechnology), and anti-p-SMAD2/3 mouse IgG (Santa Cruz Biotechnology). Then, a mixture of Alexa 488-conjugated anti-rabbit IgG (Cell Signaling) F(ab’) fragments was used for visualization of PAI-1, 4-HNE, and NOX4 proteins. A mixture of Alexa 488-conjugated anti-mouse IgG (Cell Signaling) F(ab’) fragments was used for visualization of α-SMA, COL1A1, and p-SMAD2/3 proteins. Mouse tissues were counterstained with 4′6-diamidino-2-phenylindole (DAPI). Fluorescence was visualized using an Axio inverted microscope (Carl Zeiss).

Formalin-fixed and paraffin-embedded sections of primary tumors from each group were dewaxed in an OTTIX bath (Diapath) and followed by hematoxylin (Sigma Aldrich) and eosin (Diapath). Tumor section images were shown by phase-contrast microscopy (Carl Zeiss).