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The percentage of TUNEL-positive cells in each image was determined using the 'Analyse Particles' feature of ImageJ image analysis software (Rasband, W.S., ImageJ, US National Institutes of Health, Bethesda, MD, USA, http://imagej.nih.gov/ij/, 1997 2012).
Wing size was measured in pixels using the Analyse tool in Adobe Photoshop.
The image was cropped and desitometrical analysis carried using the 'Analyse' function.
Measurements are then provided of selected areas using the "analyse particles" function in ImageJ (Fig. 3 a and b).
Branching on skeletonized binary images was measured using the Analyse Skeleton plugin (Arganda-Carreras et al, 2010).
Positively stained cells were identified and counted using the analyse particles function in Image J software (rsbweb.nih.gov).
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Data were statistically evaluated using the Analyse-it software (Version 2.03; Analyse-it Software, Leeds Leeds, UK).
The data were statistically evaluated using the Analyse-it software (Version 1.62; Analyse-it Software, Leeds Leeds, UK).
Analyses were performed in Excel 2000 (Microsoft Inc., Redmond, WA, USA) using the Analyse-It statistical add-in (Analyse-It Software Ltd, Leeds, UK) and GraphPad Prism 3.02 (GraphPad Software Inc., San Diego, CA, USA).
Selection for reproductive effort was studied using the analyses of selection gradients [48], [53].
Statistical analysis was undertaken using the Analyse-It™ add-in for Microsoft Excel.
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