Bell P B, Lindroth M, Fredriksson B A
Department of Zoology, University of Oklahoma, Norman 73019.
Scanning Microsc. 1988 Sep;2(3):1647-61.
In this tutorial we describe our methods for preparing detergent-extracted cytoskeletons for observation by high resolution scanning (SEM) and scanning transmission (STEM) electron microscopy. We both discuss the theoretical background and provide practical procedures for each of the following steps: cell culture on Formvar-coated gold grids; prefixation with aldehydes or protein crosslinking reagents (homobifunctional N-hydroxy-succinimide esters); extraction with Triton X-100 or Brij 58 detergent in microtubule stabilizing buffer; postfixation in formaldehyde and glutaraldehyde; dehydration; critical point and freeze drying; sputter coating with 1-2 nm of platinum or tungsten; and examination by SEM and both normal and inverted contrast STEM. These methods produce cytoskeletal preparations in which filaments as fine as 7 nm are preserved and can be observed by scanning electron microscopy.
在本教程中，我们描述了制备经去污剂处理的细胞骨架的方法，以便通过高分辨率扫描电子显微镜（SEM）和扫描透射电子显微镜（STEM）进行观察。我们既讨论了理论背景，又为以下每个步骤提供了实际操作程序：在福尔马林中涂覆金网格上进行细胞培养；用醛类或蛋白质交联试剂（同双功能N-羟基琥珀酰亚胺酯）进行预固定；在微管稳定缓冲液中用Triton X-100或Brij 58去污剂进行提取；在甲醛和戊二醛中进行后固定；脱水；临界点干燥和冷冻干燥；用1-2纳米的铂或钨进行溅射镀膜；以及通过SEM和正常及倒置对比度STEM进行检查。这些方法制备的细胞骨架制剂中，细至7纳米的细丝得以保存，并可通过扫描电子显微镜观察到。
