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The matrix block was saturated using recombined binary mixture live oil (C1 C7), while the fracture was filled with a sealing material to obtain a homogeneous saturation.
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For antigen retrieval (CD31 only, not necessary for VEGF) the section were microwave treated in 1 mM EDTA at pH 8 for 10 min and allowed to cool for 20 min. Endogenous biotin was saturated using a biotin blocking kit (Vector Laboratories).
Nonspecific binding sites were saturated using a blocking solution of 3%ovalbumin/2%% donkey serum in PBS.
Firstly, the samples were saturated using the blocking solution, then different couples of primary antibodies (rabbit and mouse in our case) were incubated 1 h at 37°C.
Then the sample was saturated by using deaired water.
For all patients the contrast-enhanced T1-weighted images were fat saturated using spectral saturating technique.
The 1-octanol was saturated with PBS before use.
Endogenous biotin was saturated by biotin blocking kit (Vector).
Endogenous biotin was saturated with a biotin blocking kit (Vector Laboratories).
Once the filter was saturated with RNA later®, it was sealed until further use.
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