von Schack M L, Fakan S, Villiger W, Müller M
Centre of Electron Microscopy, University of Lausanne, Switzerland.
Eur J Histochem. 1993;37(1):5-18.
A study of the fine structure of cultured mouse P815 cells as well as of mouse liver tissue after having undergone cryofixation and cryosubstitution is reported here. The P815 cells were cryofixed by a projection onto a liquid nitrogen (LN2), or liquid helium (LHe), cooled copper mirror: the liver tissue was processed (cryofixed) by high pressure freezing using a Balzers HPM 010 apparatus. No conventional chemical fixatives were used in the substitution medium which consisted of pure acetone. Embedding was carried out either in Lowicryl K11M, Lowicryl HM23, Epon or in LR White resins. The results of this study enabled us to conclude that a) one can obtain good preservation of cellular ultrastructure using different cryofixation methods and cryosubstitution without the use of chemical fixatives: b) different methods of embedding can be applied after various cryofixation techniques giving rise to slight differences in the well preserved fine structure; and c) high pressure freezing is to be recommended, in cryosubstitution studies, over that of slam freezing especially when cryofixing larger pieces of tissue which can result in a good morphology of up to 400 microns in depth.
本文报道了对经冷冻固定和冷冻置换后的培养小鼠P815细胞以及小鼠肝脏组织的精细结构研究。P815细胞通过投射到液氮（LN2）或液氦（LHe）冷却的铜镜上进行冷冻固定；肝脏组织使用Balzers HPM 010装置通过高压冷冻进行处理（冷冻固定）。置换介质由纯丙酮组成，未使用传统化学固定剂。包埋在Lowicryl K11M、Lowicryl HM23、环氧树脂或LR White树脂中进行。本研究结果使我们能够得出以下结论：a）不使用化学固定剂，采用不同的冷冻固定方法和冷冻置换能够良好保存细胞超微结构；b）在各种冷冻固定技术之后可以应用不同的包埋方法，这会在保存良好的精细结构上产生细微差异；c）在冷冻置换研究中，相比于骤冷冷冻，推荐使用高压冷冻，尤其是在冷冻固定较大组织块时，高压冷冻可使深度达400微米的组织形态良好。
