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RAGE staining was quantified as area staining positive for the brown chromagen per 100× field.
The average RAGE staining, determined as percent area staining positive for the brown chromogen per 100× field, for the diabetic left limbs (21.3 ± 6.2%) was significantly greater than that for the contralateral right limb (5.6 ± 4.3%; P = 0.02) or non-diabetic left limbs (7.6 ± 5.6%; P = 0.04).
All brown staining areas were counted for each of the three sections for both the left and right anterior tibialis muscles for each experiment, and averaged RAGE and lectin staining were quantified as area staining positive for the brown chromogen per 100× field.
Hearts were considered positive for RV fibrosis if they contained an area staining positive for Masson's trichrome which extended from the inner aspect to the outer aspect of the RV free wall.
Images were analyzed for percentage area staining positive for fibronectin using image analysis software (Metamorph).
CD34 staining accounted for less than 10% of the area staining for vimentin.
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Staining intensity was scored as follows: (1) <10% of tumour area stained; (2) 11 25% stained; (3) 26–50% stained; and (4) ⩾51% stained.
Values are given as percent of area stained (mean ± SD).
Necrosis was defined as an area stained with haematoxylin that originated from proteins released from the cytoplasm of dead cells.
Image-Pro 6.2 software was used to calculate the area stained with nuclear fast red, and the area stained with Alcian blue.
Calculation of the proportional area stained was then determined using image analysis, as described [54].
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