基于块染技术的电镜生物样本免切片染色制备方法探究OA

The appropriate block staining method is crucial for ensuring the quality of biological electron microscopy imaging.Based on conventional biological sample block staining,this study systematically investigated 13 direct block staining methods for biological samples that can be observed with a transmission electron microscope(TEM)before embedding,using mouse kidneys as a model.The results showed that the observation of ultrastructure was affected by different staining reagents and staining sequences,compared with the conventional uranium block staining method.Among them,the samples prepared by the osmium tetroxide-thiocarbohydrazide-osmium tetroxide(OTO method),the osmium tetroxide-tannic acid-tannic acid-osmium tetroxide(OTTO method),and the potassium ferrocyanide-osmium tetroxide-thiocarbohydrazide-osmium tetroxide(ROTO method)showed improved structural clarity and image contrast of organelles such as mitochondria.The staining sequence of heavy metals lead and uranium was found to affect the clarity of mitochondrial cristae structure.The optimized potassium ferrocyanide-osmium tetroxide-tannic acid-tannic acid-osmium tetroxide(ROT(T)O method),when combined with lead and uranium double block staining,not only increased the sample contrast,but also more effectively maintained the cytoskeleton and cell membrane structure,thereby achieving the best imaging effect.