Alexa 488 conjugated anti 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).

Immunohistochemistry with Doublecortin (DCX) was conducted as previously reported [12 (link)]. Briefly, after behavioral testing, half of the brains were fixed by 4% paraformaldehyde in PBS, pH7.4 at 4°C for 48 hours. They were transferred to 30% sucrose and then cut with a Microm freezing microtome at a thickness of 20 μm and stained with progenitor neuronal markers DCX (anti-rabbit, 1:100, Santa Cruz) overnight at 4°C. After washing with PBS, the sections were incubated with Alexa 488-conjugated anti-rabbit IgG (1:500, Cell Signaling) followed by counter-staining with DAPI and then cover-slipped with anti-fade mount solution (Molecular Probes). Images were acquired with a Zeiss LSM PASCAL confocal microscope.

Cells were fixed with 4% paraformaldehyde (PFA) (Santa Cruz Biotechnology) in PBS for 15 min at room temperature, permeabilized with 0.3% Triton X-100 (AppliChem) in PBS for 15 min, and then incubated with 2% bovine serum albumin (BSA) (Sigma-Aldrich) in PBS for 1 h. Fixed cells were then incubated overnight at 4 °C with anti-human NF-κB RelA (Santa Cruz Biotechnology), incubated for 1 h with Alexa 488-conjugated anti-rabbit IgG (Cell Signaling Technology), and finally mounted in Vectashield (Vector Laboratories) with DAPI to counterstain the nuclei.
Confocal images were acquired using LSM 800 laser scanning confocal microscope (Zeiss) using an apochromat × 40/1.4 oil objective (Zeiss) at the Bioimaging Core Facility, Faculty of Medicine, University of Geneva. Image analysis was performed with ImageJ software.