Alexa 488 conjugated anti mouse 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).

OCT-embedded cryo kidney sections (4 μm) were fixed in acetone for 20 min and blocked with 10% BSA for 1 h. Sections were then incubated with rabbit anti-mouse C3 (Cat: PA1-40,288), APC conjugated anti-mouse CD4 (Cat: 17–0042-82), Alexa-488 conjugated anti-sheep IgG (Cat: A11015), or Alexa-488 conjugated anti-mouse IgG (Cat: A21202), for 1 h at room temperature, followed by Rhodamine-conjugated anti-rabbit (Cell Signaling Technology Beverly, MA. Cat: R6394, 1:100 dilution) for C3 secondary antibodies staining for 1 h. All primary Abs were purchased from Life Technologies (Carlsbad, CA, 1:100 dilution). Slides were covered with Fluoromount-G mounting medium (SouthernBiotech, Birmingham, AL) after three times washing with PBS. Images were acquired with a Leica DMi8 fluorescent microscope (Buffalo Grove, IL) and analyzed with the LAX S software (Leica Microsystems Inc.). CD4⁺ T cell numbers were counted in four (200 ×) low-power fields in each section. Sections from at least 4 mice were counted and analyzed.