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Each result was corrected by subtraction of mean background intensities for each channel.
Metrics for all sample hybridizations including scaling factors, mean background intensities, and percent present calls have been provided (see Additional File 1).
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The mean background intensity from control and anti-E-selectin-targeted animals was subtracted.
Labeling was considered positive when the intensity of the fluorescent signal exceeded the 95% confidence interval of the mean background intensity.
From these scans, we obtained a relative transmissionaTrel(red dashed line in Figure 1) by normalizing the measured intensity Im with the mean background intensity Ib from regions without particles: Trel=Im/Ib.
A vessel was considered positive if mean VCAM-1 intensity was higher than mean background intensity, whereas it was considered negative if mean VCAM-1 intensity was below.
Global background subtraction was performed by subtracting the mean background intensity plus two standard deviations from the signal of all experimental spots.
The ratios were calculated after subtracting the mean background intensity.
We adjusted mean signal intensity for local background by subtracting the mean background intensity.
Additionally, the mean background intensity was determined from all pixels outside the spots.
Because background intensity varied between slides, the mean background intensity was subtracted from the mean intensity for each axonal measurement.
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