Finally, fluorescent image montages of the EF5/Cy3 staining were quantified by determining the mean pixel intensity of each image (range: 0 255).
The level of total BODIPY-TMR-CGP binding was quantified by determining the average pixel intensities for each image, which was then plotted against the BODIPY-TMR-CGP concentrations used, to obtain a saturation binding plot.
For in vivo staining, tumours were cut in 6 -μm sections using a cryostat microtome, mounted on gelatinised slides, processed and analysed by determining the mean pixels intensities of random microscopic fields by using the ImageJ software (NIH).
Relative fluorescence was calculated by determining the intensity per pixel for the various emission settings, by measuring the mean intensity of representative areas of nucleus or cytoplasm and normalised by dividing by the attenuation, as above.
Quantification of immunoblot band optical density was calculated by determining the sum of the pixel intensity for each band subtracted by the average background.
Quantification was accomplished by determining the number of pixels in the negative images.
BDA-positive fibre densities were calculated using ImageJ software by determining the number of pixels above a set intensity threshold.
This conversion was accomplished by determining the number of pixels between two lines of the image of a standard slide having 10 μm divisions.
22 The sections were digitized and the area of BDA-labeled axons was quantified by determining the number of pixels above the intensity gray-scale threshold using ImageJ (NIH) as described previously.
The percentage of attached bacteria was calculated by determining the ratio of object pixels to the total number of pixels.
