Lima-Faria João Marcos de, Guimarães Lucas Nunes, Silva Victória Costa da, Souza Iara da Costa, Fernandes Marisa Narciso, Martinez Diego Stéfani Teodoro, Sabóia-Morais Simone Maria Teixeira de
Laboratory of Cellular Behavior, Institute of Biological Sciences, Federal University of Goiás, Brazil.
Dept. Physiological Science-CCBS, Federal University of São Carlos, Brazil.
MethodsX. 2022 Jun 21;9:101769. doi: 10.1016/j.mex.2022.101769. eCollection 2022.
Toxicity evaluations involve the analysis of multiple biomarkers. In this study, the liver, target organ analyzed by treatments with iron concentrations, indicated the accumulation of lipids as a response. Considering that the distribution of lipids in an organ is directly related to the induction of inflammatory processes by aquatic contaminants, this study proposes to carry out an integrative investigation of the behavior and the distribution of lipids in the liver tissue. Techniques of light and electron microscopy were performed in order to propose a new way of assessing and quantifying the distribution of lipid droplets, also presenting methodological alternatives that can be chosen by the reader according to the interests and resources available. Thus, it is assumed that the method begins with the fixation of the liver with Glutaraldehyde 2,5% in PBS 0,1 M and continues with post fixation with osmium tretoxide 1%, which marks lipids. For this proposition, two inclusion methodologies were performed to histological analyses in Historesin and ultrastructural analyses in EMBeed 812. For light microscopy (LM) analyses, cuts were obtained with 2,5 micrometers thickness, which were stained with (1) Mayers hematoxylin and (2) toluidine blue. The images obtained were processed in software Image J Fiji to evidence the lipid distribution in liver.•Cytological reactions with osmium tetroxide constitute low complexity methods that allow the optimization of the localization, identification and quantification of lipid droplets in the liver tissue when analyzed under the conventional light microscope.•Samples included in EMBeed 812 resin commonly used in Transmission Electron Microscopy can be analyzed by SEM-BEC, as complementary analyses for the detection of lipids.•Using SEM-BEC and conventional light microscopy, it is possible to quantify the area occupied by lipid droplets using Image J Fiji software, as these are contrasted due to the reaction with osmium tetroxide.
毒性评估涉及多种生物标志物的分析。在本研究中，肝脏作为通过不同铁浓度处理进行分析的靶器官，显示出脂质积累作为一种反应。鉴于器官中脂质的分布与水生污染物诱导的炎症过程直接相关，本研究建议对肝脏组织中脂质的行为和分布进行综合研究。进行了光学显微镜和电子显微镜技术，以提出一种评估和量化脂滴分布的新方法，还提供了可供读者根据自身兴趣和可用资源选择的方法替代方案。因此，假定该方法首先用0.1M PBS中的2.5%戊二醛固定肝脏，然后用1%的锇酸进行后固定，锇酸可标记脂质。为此，进行了两种包埋方法用于组织学分析（在Historesin中）和超微结构分析（在EMBeed 812中）。对于光学显微镜（LM）分析，获得厚度为2.5微米的切片，并用（1）迈耶苏木精和（2）甲苯胺蓝染色。获得的图像在Image J Fiji软件中进行处理，以显示肝脏中的脂质分布。•四氧化锇的细胞学反应构成低复杂度方法，当在传统光学显微镜下分析时，可优化肝脏组织中脂滴的定位、识别和量化。•包含在常用于透射电子显微镜的EMBeed 812树脂中的样本可通过扫描电子显微镜-背散射电子成像（SEM-BEC）进行分析，作为脂质检测的补充分析。•使用SEM-BEC和传统光学显微镜，利用Image J Fiji软件可以量化脂滴所占面积，因为这些脂滴由于与四氧化锇的反应而形成反差。
