Image pro premier software

Islets were quantified by Image-pro Premier software (Media Cybernetics, Rockville, MD). For each islet, the percentage of the positive area (or positive staining, defined by Image-pro Premier software) within the islet boundary was assessed and the differences between the groups and within the regions of each section were presented as the mean of analyzed islets (mean±SD).

These were as reported previously^{30 (link)}. At sacrifice, the upper portion of the heart and proximal aorta was obtained, embedded in OCT compound, and stored at −70 °C. Serial 10 μm thick cryosections of the aorta, beginning at the aortic root, were collected on poly-D-lysine–coated slides. These sections were stained with Oil Red O and hematoxylin. The images were taken using a Nikon Eclips microscope. Ten sections at 120 μM intervals were counted from each mouse. ImagePro premier software (Media Cybernetics, MD, USA) was used to count the lesion area. This study adhered to the guidelines for experimental atherosclerosis studies as described in the American Heart Association Statement. Circulating leukocyte levels were examined using a HemaTrue analyzer.

Samples on slides were prepared as described above and then imaged using a Zeiss 780 laser scanning confocal microscope. Three-dimensional eight-bit image stacks of the various fluorescent signals were acquired sequentially using a 63X (1.4 na) objective using a step size of 0.3 μm between images. These stacks of images were then projected into single stacks for two-dimensional analysis by using a maximum intensity projection macro in the Zeiss Zen software (Zeiss Inc., Thornwood, NY) or a similar macro in ImageJ (National Institutes of Health, Bethesda, MD). For further analysis of the fluorescent signals per cell, the images were imported into Image-Pro Premier software (Media Cybernetics, Rockville, MD). Here all labeled cells were autotraced using a region of interest module in the software based on their mCherry signal. These outlines were used to extract and/or score the total area and fluorescence signal of the BODIPY 493/503 signal within the autotraced cell outlines.^{11 (link)} A defined threshold above background and autofluorescence was used for all BODIPY 493/503 relevant signal in the cells, and this range was 75–256 for the eight-bit images. These values of total BODIPY 493/503 signal per cell above background were then imported into Excel for further processing where we obtained integrated density values per cell (area × mean fluorescence intensity).