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\nThe use of the \"largest contour\" approach was deemed more reliable than the \"surface area\" or \"volume\" methods in several previous studies, with the use of a fixed threshold of at least 75% of the maximum intensity of the entire image [[@B7],[@B9],[@B10]]. Although a fixed threshold of 75% of the maximum intensity had been adopted in the present study, there was a debate whether such threshold was a suitable value for use with the mouse cross-sections. The data suggest that the results generated using different fixed thresholds are not equivalent. The best way to overcome this issue is to generate a suitable threshold with which to evaluate the average value of the median signal for each mouse in the cohort of a particular experiment. This can be achieved by setting the threshold to 0.2 times the minimum intensity value of the ROI, as the minimum intensity value of the ROI represents the lower limit of the background signal, which is impossible to generate within the tissue-depositing area.
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