The objective of the present study was to develop a stain permitting automated quantification of myocellular lipid depositions in skeletal muscle sections together with immunolocalisation of other myocellular constituents by fluorescence microscopy. Lipid droplets were detected in skeletal muscle by oil red O (ORO). Conventional ORO was modified to diminish background staining, prevent crystallisation of ORO and to optimise lipid retention in cryosections. These modifications resulted in a punctate staining of lipid droplets, rather than the somewhat diffuse staining by conventional ORO. Small cavities in muscle sections (like the lumen of small blood vessels) lack ORO when using the protocol presented here. In addition a staining protocol is presented combining ORO with immunofluorescence. This combination permits multiple staining studies in the same section. Thus, lipid droplets can be studied together with immunolabelling of proteins involved in lipid handling and metabolism. This will extend our knowledge on the subcellular localisation of lipid handling proteins (i.e. enzymes and fatty acid transporting proteins) in relation to the localisation of lipid depositions. In conclusion, the protocol presented here permits examination of ORO-stained lipid droplets in skeletal muscle sections together with multiple staining of other immunodetectable proteins present in skeletal muscle by quantitative fluorescence microscopy.

中文翻译：

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油红O染色的优化可与免疫荧光和脂质的自动定量相结合。

本研究的目的是开发一种染色剂，该染色剂可通过荧光显微镜自动定量骨骼肌切片中的肌细胞脂质沉积以及其他肌细胞成分的免疫定位。通过油红O（ORO）在骨骼肌中检测到脂质滴。对常规ORO进行了改良，以减少背景染色，防止ORO结晶并优化冷冻切片中的脂质保留。这些修饰导致脂质液滴的点状染色，而不是常规ORO的有点弥散性染色。使用此处介绍的方案时，肌肉部分的小腔（如小血管的内腔）缺少ORO。此外，提出了将ORO与免疫荧光结合的染色方案。这种组合允许在同一部分进行多次染色研究。因此，可以将脂质滴与参与脂质处理和代谢的蛋白质的免疫标记一起进行研究。这将扩展我们对与脂质沉积的定位有关的脂质处理蛋白（即酶和脂肪酸转运蛋白）的亚细胞定位的认识。总之，这里介绍的方案允许通过定量荧光显微镜检查骨骼肌切片中ORO染色的脂质滴以及骨骼肌中存在的其他免疫可检测蛋白的多重染色。这将扩展我们对与脂质沉积的定位有关的脂质处理蛋白（即酶和脂肪酸转运蛋白）的亚细胞定位的认识。总之，这里介绍的方案允许通过定量荧光显微镜检查骨骼肌切片中ORO染色的脂质滴以及骨骼肌中存在的其他免疫可检测蛋白的多重染色。这将扩展我们对与脂质沉积的定位有关的脂质处理蛋白（即酶和脂肪酸转运蛋白）的亚细胞定位的认识。总之，这里介绍的方案允许通过定量荧光显微镜检查骨骼肌切片中ORO染色的脂质滴以及骨骼肌中存在的其他免疫检测蛋白的多重染色。