Stained femurs on slides were scanned using an Aperio CS slide scanning system with a Spectrum Plus information management system (Aperio Technologies, Inc., Vista, CA). The ImageScope program (Aperio) was used to quantify positive staining in two regions of marrow (distal middle and middle) using the Positive Pixel Count algorithm (Aperio). Positive pixels were determined by a range of hues, saturations, and intensities of NovaRED. Negative controls for all targets were analyzed to ensure that the algorithm detected minimal positive pixels. Adiposity of the bone marrow was assessed in a representative region within the middle distal femur that spanned from the medial cortex to the lateral cortex and constituted an area between 1 and 1.5 mm2. Adipose remains unstained and is thus not considered positive or negative by the algorithm. Therefore, we subtracted the area of positive and negative pixilation from the total area of the selected region. We were careful to select regions devoid of nonstained artifact, for example, tears in the tissue resulting from processing.
Cs slide scanning system
Manufactured by Leica
The CS slide scanning system is a high-performance digital slide scanner designed for efficient scanning of histological and cytological samples. It captures high-resolution digital images of glass slides for use in various laboratory and research applications.
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2 protocols using cs slide scanning system
1
Quantifying Bone Marrow Adiposity
2
Retinal Vascular Density Quantification
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For relative CD31 þ blood vessel density, at least 10 images (20Â objective) were taken from each animal eye (two images for the central retina, four images for the mid retina, and four images for peripheral retina) using a Zeiss Axio Imager Upright microscope. The relative CD31 þ blood vessel density in the entire image (13.5 Â 10 cm) was measured by determining the frequency of positive immunolabeled CD31 þ vascular structures coinciding with the intersection of 100 intersection points with a diameter of 0.8 cm in a 13.5 Â 10-cm frame superimposed over the image, as reported previously. 16, 17 Trypsin Digest Preparation and Quantitative Analysis of Relative Acellular Capillary Density Trypsin digests of retinal whole mounts were prepared to permit examination of the frequency of acellular capillary density using the method described by Kuwabara and Cogan, 18 with minor modifications. Retinas were scanned using the Aperio CS slide scanning system with Spectrum Plus information management system (Aperio Technologies Inc., Vista, CA). A total of 10 to 15 random, nonoverlapping fields of retina were imaged (100,000 mm 2 ). Acellular capillaries >50 mm in length were counted from each image for each retina and expressed relative to control.
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