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Vectastin elite abc reagent

Manufactured by Vector Laboratories
Sourced in United States

Vectastin Elite ABC Reagent is a ready-to-use avidin-biotin complex (ABC) reagent designed for immunohistochemical and immunocytochemical staining procedures. It provides a sensitive detection system for locating target antigens in tissue sections or cell preparations.

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2 protocols using vectastin elite abc reagent

1

Protein Expression Evaluation in Cell Lines

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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.
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2

Immunofluorescence of Cultured Cells

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Exponentially growing cell line cells 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 70% confluent. The cells were fixed with buffered paraformaldehyde at room temperature, incubated with 1% H2O2 in methanol to block endogenous peroxidase and again washed twice with buffer solution. Subsequently, the cell cultures were then covered with normal horse serum for 30 min at room temperature and incubated with either anti-mouse or anti-goat monoclonal or polyclonal antibodies: ERα (mouse, sc-8002), Neu (rabbit, sc-284) and H-Ras (mouse, sc-29) (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 1:500 dilution overnight at 4°C and then incubated for 45 min with diluted biotinylated secondary antibody solution (Vector Laboratories Inc., Burlingame, CA, USA) and Vectastin Elite ABC reagent (Vector Laboratories Inc.) was used. The experiments were repeated twice in cells with identical passages in vitro.
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