Brain tissue sections were stained for blood vessel marker CD31 (AF3628, R&D Systems), VCAM‐1 (1510‐14, Southern Biotech), and the macrophage/microglia marker Iba‐1 (ab5076, Abcam, Cambridge, UK), as described previously.
45 (link),
46 (link),
47 (link) Secondary antibodies used were biotinylated horse anti‐goat IgG (BA‐9500, Vector Laboratories, Peterborough, UK) and biotinylated goat anti‐rat IgG (BA‐9401, Vector Laboratories). Sections were also stained for the angiogenic marker integrin αvβ3 (ab78289, Abcam) using Mouse on Mouse Basic Kit (BMK‐2202, Vector Laboratories) in accordance with the protocol described by the manufacturer.
Microvessel density (CD31 positive area fraction) was quantified for the area encompassing the metastatic foci in the injected striatum, and also for the contralateral striatum, as the percentage of area covered using the ‘Positive Pixel Count 2004‐08‐11’ algorithm in ImageScope (Leica Biosystems). The parameters used were moderately stained pixel intensity, between 202 and 185, and strongly stained pixel intensity, less than 10.
45 (link),
46 (link),
47 (link) Secondary antibodies used were biotinylated horse anti‐goat IgG (BA‐9500, Vector Laboratories, Peterborough, UK) and biotinylated goat anti‐rat IgG (BA‐9401, Vector Laboratories). Sections were also stained for the angiogenic marker integrin αvβ3 (ab78289, Abcam) using Mouse on Mouse Basic Kit (BMK‐2202, Vector Laboratories) in accordance with the protocol described by the manufacturer.
Microvessel density (CD31 positive area fraction) was quantified for the area encompassing the metastatic foci in the injected striatum, and also for the contralateral striatum, as the percentage of area covered using the ‘Positive Pixel Count 2004‐08‐11’ algorithm in ImageScope (Leica Biosystems). The parameters used were moderately stained pixel intensity, between 202 and 185, and strongly stained pixel intensity, less than 10.
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