Biotinylated secondary antibody solution

For bright-field microscopy, free-floating sections were quenched by incubation in a mixture of 3% H₂O₂ and 10% methanol in tris-buffered saline (pH 7.6). Nonspecific binding sites were blocked by incubation in 5% normal serum. Samples were kept overnight at room temperature in a solution containing the primary antibody: rabbit anti–h-αS (1:50,000; ab138501, Abcam), rabbit anti-RFP (1:20,000; 600-401-379, Rockland), mouse anti–Cre recombinase (1:4000; MAB3120, Millipore), rabbit anti–c-fos (1:2000; 2250, Cell Signaling Technology), and rabbit anti-SOD2 (1:5000; ADI-SOD-110, Enzo Life Sciences). Sections were rinsed and incubated in biotinylated secondary antibody solution (1:200; Vector Laboratories). Following treatment with avidin-biotin–horseradish peroxidase complex (ABC Elite kit, Vector Laboratories), color reaction was developed using a 3,3′-diaminobenzidine kit with or without nickel (Vector Laboratories). Sections were mounted on coated slides, coverslipped with Depex (Sigma-Aldrich), and imaged using a Zeiss Observer.Z1 Microscope (Carl Zeiss) equipped with a motorized stage and AxioCam MRm camera (Carl Zeiss). For bright-field density measurements, slides containing MO sections were scanned (AxioScan.Z1, Carl Zeiss). The DMnX was delineated on three equally spaced sections, and integrated density values were obtained using Fiji (ImageJ version 2.1.0/1.53c).

Protein expression was evaluated as previously described (22 (link)–24 (link)). Exponentially growing cell lines were plated on a glass chamber slide (Nunc Inc., Naperville, IL, USA) at a density of 1×10⁴ cells/ml of medium and allowed to grow for 2–3 days until they reached 70% confluence (21 (link)). The cells were fixed with buffered paraformaldehyde at room temperature, incubated with 1% H₂O₂ in methanol to block endogenous peroxidase and washed twice with buffer solution. Cell cultures were subsequently covered with normal horse serum for 30 min at RT and incubated with anti-rabbit monoclonal antibody (vimentin: C-20, sc 7557 and Notch 4: C-19, sc 8644) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a 1:500 dilution at 4ºC overnight, and then incubated for 45 min with diluted biotinylated secondary antibody solution (Vector Laboratories, Burlingame, CA, USA) and Vectastin Elite ABC Reagent (Vector Laboratories). The experiments were repeated three times in cells with identical passages in vitro. The number of immune-reactive cells (50 cells/field) was counted in several randomly selected microscopy fields (×400) per sample using an optical microscope (C×31; Olympus Corporation, Tokyo, Japan). Ten fields were counted for each cell line.