Anti 4 hne rabbit igg

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).

For in vitro assay, cells were seeded on cover glass in 6 cm plates and were incubated for 24 h for attachment. The cells were incubated with serum-reduced (0.2% FBS) medium for 16 h for starvation and irradiated with 10 Gy with or without 30-min pretreatment with 100 nM vactosertib. After 24 h, cells were fixed with 4% formaldehyde solution (pH 7.4) and were blocked in 5% BSA with normal serum in 0.1% Triton 100X. Then, cells were incubated with primary and secondary antibodies. Anti-4-HNE rabbit IgG (Abcam) and Alexa 488-conjugated anti-rabbit IgG (Cell Signaling) F(ab’) fragments were used as primary and secondary antibody, respectively. Cells were counterstained with DAPI. Fluorescence was visualized using an Axio inverted microscope (Carl Zeiss).