![]() As seen in Figure 3, bright-field and DAPI images were taken to identify total tissue. The processing of LipidTox images was determined using visual threshold levels, as different sections had variable intensities of fluorescence. When thresholding and segmentation are complete, the result is 2 separate images. The lipids are removed during tissue processing however, their absence is seen as intracellular white space and is the basis of the estimation of lipid content. The white space is either background or space previously occupied by lipid. Comparing the binary, Otsu processed images to the 8-bit, original H&E stain, there is a clear visual match between what is seen as white and black space, and what is stained as tissue and nuclei. With the grayscale H&E images, there is a clear distinction between background (white) and foreground (pink) seen in the presence of 2 peaks on the histogram of each image, which allows the threshold to easily separate the 2 rapidly. ![]() This thresholding algorithm assumes a bimodal distribution of foreground and background in an image to convert the gray scale into a binary result (15). The second pathway applies an Otsu threshold to the image. This method allows for reproducible and comparative lipid assessment in complex adipose tissues with multilocular properties. The protocol is low cost, has high inter-rater reliability, and results are comparable to quantification using lipid stains. We validated a standardized method to estimate lipid content within thermogenic adipose tissue (BAT and PVAT) that can be performed on routine histological slides stained with hematoxylin/eosin (H&E). These methods are more time consuming than routine histology, and require cryosectioning, expense of the reagent, and fluorescence microscopy. Staining methods include oil red O or lipid probes such as BODIPY (4,4-difluoro-3a,4a-diaza-s-indacene) or LipidTOX. The multilocular phenotype of thermogenic adipose tissue poses a challenge for morphometric quantification using current methods. In the mouse, BAT is found as interscapular lobes underneath subcutaneous WAT located on the back. PVAT is found adjacent to large vessels in the body, specifically around the aorta where it can be collected with relative ease. PVAT also releases factors, which can differ under varying phenotypes that have local effects on the vasculature. BAT and PVAT in the mouse are thermogenic adipose tissues. Multilocular thermogenic adipocytes are known for their fat-burning phenotype, where mitochondrial uncoupling from ATP generation results in lipid breakdown to release heat. Often these droplets are quite small and cannot be easily measured. In contrast to the unilocular adipocytes used for lipid storage in WAT, brown adipose tissue (BAT) and mouse PVAT are filled with multilocular adipocytes, where numerous lipid droplets of varying size occupy the cytoplasm, making cell borders difficult to distinguish. Results indicate that lipid content can be estimated within mouse PVAT in a quantitative and reproducible manner, and shows correlation with previously studied molecular and physiological measures. We used our method to analyze perivascular adipose tissue (PVAT) from C57BL/6 mice on a normal chow diet, compared to calorie restriction or a high fat diet, where lipid storage phenotypes are known. The method was compared to direct lipid staining of adipose tissue, with comparable results. This method, using FIJI analysis of hematoxylin/eosin stained sections, was highly objective and highly reproducible, with ∼99% inter-rater reliability. ![]() We developed a simple, standardized method to quantify lipid content of mouse thermogenic adipose tissue. However, thermogenic adipose tissue has multilocular adipocytes, making it difficult to distinguish adipocyte cell borders and to analyze lipid proportion using existing methods. Quantification of adipocyte size and number is routinely performed for white adipose tissues using existing image analysis software. ![]()
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