Anti fibronectin

The hippocampi of Tg2576 mice (n = 3/group) were sonicated in Ripa lysis buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Igepal, 50 mM NaF, 1 mM NaVO₃) containing a protease inhibitor cocktail. Proteins (15–45 μg) were separated by gel electrophoresis on 3–8% Tris-acetate or 10–20% Tris-Tricine gels (Life Technologies), transferred to nitrocellulose membranes and incubated with the following antibodies: anti-APP CTF (1:1000, BioLegend, London, UK), anti-Aβ1-16 (clone 6E10, 1:500, BioLegend), anti-nicastrin (1:500, New England Biolabs, Hitchin, UK), anti-β-secretase-1 (BACE, 1:750, New England Biolabs), anti-low-density receptor-related protein-1 (LRP-1, 1:2500, Insight Biotechnology, Wembley, UK), anti-IDE (1:750, Abcam, Cambridge, UK), anti-neprilysin (1:500, Abcam, Cambridge, UK), anti-fibronectin (1:750, AbD Serotec) and anti-collagen IV (1:500, Sigma Aldrich). Membranes were stripped and reprobed with anti-glyceraldehyde-3-PDH (GAPDH) antibody (1:50,000; Sigma Aldrich) to ensure equal protein loading. Immunoblots were repeated twice per antibody and quantified by densitometry using Image J software. Means ± S.E.M. were calculated as an optical density ratio of protein levels normalized to GAPDH levels.

Mice (n = 5/group) were perfused intracardially with 0.01M PBS, followed by 4% paraformaldehyde, brains removed and post-fixed overnight. For enzyme-linked immunohistochemistry, brain tissue sections were washed in 0.01M PBS, treated with formic acid (70%, 30 sec) and incubated overnight at 4 °C with antibodies against human anti-Aβ40 (1:250, Merck Millipore, Watford, UK) and anti-Aβ42 (1:250, Merck Millipore). Sections were washed in 0.01M PBS, incubated with biotinylated anti-rabbit (1:400, Vector Labs, Peterborough, UK) and developed using glucose oxidase enhancement with DAB as chromagen. For fluorescent immunohistochemistry, tissue sections were washed in 0.01M PBS, treated with 1 mg/mL pepsin in 0.2N HCl (45 sec, 37 °C) and incubated overnight at 4 °C with anti-fibronectin (1:400, AbD Serotec, Kidlington, UK). Sections were washed in 0.01M PBS, incubated 2 hrs with AlexaFluor 488-conjugated anti-rabbit (1:200; Life Technologies) and coverslipped with mowiol. Images were captured on a Nikon microscope and exported to Photoshop CS. For quantification of percentage area covered by staining, micrographs were converted to binary images, thresholded to eliminate background staining and evaluated by densitometry using Image J software (NIH, Maryland, USA).