Institute of Pharmacology & Experimental Therapeutics & Coimbra Institute for Clinical and Biomedical Research (iCBR), Faculty of Medicine, University of Coimbra; Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra; Clinical Academic Center of Coimbra (CACC); Polytechnic Institute of Coimbra, Coimbra Health School.
Institute of Pharmacology & Experimental Therapeutics & Coimbra Institute for Clinical and Biomedical Research (iCBR), Faculty of Medicine, University of Coimbra; Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra; Clinical Academic Center of Coimbra (CACC).
J Vis Exp. 2023 Jun 2(196). doi: 10.3791/65206.
Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing dysfunction caused by free fatty acids (FAs). The liver, given its critical role in the body's fat metabolism, is persistently threatened by the intracellular accumulation of LDs in the form of both microvesicular and macrovesicular hepatic steatosis. The histologic characterization of LDs is typically based on lipid-soluble diazo dyes, such as Oil Red O (ORO) staining, but a number of disadvantages consistently hamper the use of this analysis with liver specimens. More recently, lipophilic fluorophores 493/503 have become popular for visualizing and locating LDs due to their rapid uptake and accumulation into the neutral lipid droplet core. Even though most applications are well-described in cell cultures, there is less evidence demonstrating the reliable use of lipophilic fluorophore probes as an LD imaging tool in tissue samples. Herein, we propose an optimized boron dipyrromethene (BODIPY) 493/503-based protocol for the evaluation of LDs in liver specimens from an animal model of high-fat diet (HFD)-induced hepatic steatosis. This protocol covers liver sample preparation, tissue sectioning, BODIPY 493/503 staining, image acquisition, and data analysis. We demonstrate an increased number, intensity, area ratio, and diameter of hepatic LDs upon HFD feeding. Using orthogonal projections and 3D reconstructions, it was possible to observe the full content of neutral lipids in the LD core, which appeared as nearly spherical droplets. Moreover, with the fluorophore BODIPY 493/503, we were able to distinguish microvesicles (1 µm < d ≤ 3 µm), intermediate vesicles (3 µm < d ≤ 9 µm), and macrovesicles (d > 9 µm), allowing the successful discrimination of microvesicular and macrovesicular steatosis. Overall, this BODIPY 493/503 fluorescence-based protocol is a reliable and simple tool for hepatic LD characterization and may represent a complementary approach to the classical histological protocols.
脂滴（LDs）是一种专门的细胞器，介导脂质储存，在抑制脂毒性和防止游离脂肪酸（FAs）引起的功能障碍方面发挥着非常重要的作用。肝脏在体内脂肪代谢中起着至关重要的作用，一直受到以微泡性和大泡性肝脂肪变性形式存在的细胞内 LDs 积累的威胁。LDs 的组织学特征通常基于脂溶性重氮染料，如油红 O（ORO）染色，但许多缺点一直阻碍着这种分析方法在肝标本中的应用。最近，亲脂性荧光团 493/503 由于其快速摄取和积累到中性脂质滴核心中，已成为用于可视化和定位 LDs 的流行方法。尽管大多数应用在细胞培养中得到了很好的描述，但在组织样本中，作为 LD 成像工具，亲脂性荧光探针的可靠应用证据较少。在这里，我们提出了一种基于硼二吡咯甲川（BODIPY）493/503 的优化方案，用于评估高脂肪饮食（HFD）诱导的肝脂肪变性动物模型中肝标本中的 LDs。该方案涵盖了肝样本制备、组织切片、BODIPY 493/503 染色、图像采集和数据分析。我们证明了 HFD 喂养后肝 LDs 的数量、强度、面积比和直径增加。使用正交投影和 3D 重建，可以观察到 LD 核心中中性脂质的全部含量，这些脂质呈近球形液滴。此外，使用荧光团 BODIPY 493/503，我们能够区分微泡（1 µm < d ≤ 3 µm）、中间泡（3 µm < d ≤ 9 µm）和大泡（d > 9 µm），从而成功区分微泡性和大泡性脂肪变性。总的来说，这种基于 BODIPY 493/503 荧光的方案是一种可靠且简单的肝 LD 特征描述工具，可能是对经典组织学方案的补充。
