Aged control mouse ST-3xTg-AD and LT-3xTg-AD brain sections (50 μm) were pre-treated with formic acid (20%) for 20 min for β-amyloid detection. Then, they were treated for 20 minutes in 0.1 M PB (methanol (1:1) and 1% hydrogen peroxide) and incubated for 1 h in 0.1 M PB with 1% BSA and 0.3% Triton X-100. The slices were then incubated with the primary antibody, anti-β-amyloid 1-16 (6E10) (SIGNET), overnight at 4°C in 0.1 M PB with 0.3% BSA and 0.3% Triton X-100. The slices were incubated with biotinylated mouse secondary antibody and then with the ABC-HRP complex (Pierce Biotechnology). Diaminobenzidine (DAB) was used for detection. The tissue was dehydrated, covered with mounting solution and observed under an Eclipse E200 optical microscope (Nikon). The number of extracellular β-amyloid plaques was quantified in the CA1 area of the hippocampus.
Abc hrp complex
Manufactured by Thermo Fisher Scientific
The ABC-HRP complex is a detection reagent used in various immunohistochemistry and immunocytochemistry applications. It consists of an avidin-biotin complex (ABC) coupled with horseradish peroxidase (HRP) enzyme. The complex binds to biotinylated secondary antibodies, amplifying the signal for enhanced visualization of target antigens.
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2 protocols using abc hrp complex
1
Quantifying Extracellular Amyloid Plaques in Aged Transgenic Mouse Brains
2
Immunohistochemical Analysis of Amyloid-Beta and BACE1
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Human brain sections (50 μm) were pre-treated with 10 Mm Tris, pH 6.0, at 85°C for 5 min. The slices were treated with formic acid (20%) for 20 min to detect the β-amyloid protein. The mouse (50 μm coronal sections) and human brain sections were “treated for 20 min in 0.1 M PB:methanol (1:1) with 1% hydrogen peroxide and then incubated for 1 h in 0.1 M PB with 1% BSA and 0.3% Triton X-100. Slices were then incubated with primary antibodies, mouse anti-human amyloid beta protein (1:250, SIGNET) and rabbit anti-BACE1 C-terminal (485–501; 1:100, Calbiochem), overnight at 4°C in 0.1 M PB with 0.3% BSA and 0.3% Triton X-100. The slices were incubated with a biotinylated mouse secondary antibody and then incubated with ABC-HRP complex (Pierce Biotechnology) for 2 h. Diaminobenzidine (DAB) was used to develop the staining. The tissues were dehydrated, covered with mounting solution and observed on an Eclipse E200 optical microscope (Nikon)”.
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