Effects of acetone, methanol, or paraformaldehyde on cellular structure, visualized by reflection contrast microscopy and transmission and scanning electron microscopy
Effects of acetone, methanol, or paraformaldehyde on cellular structure, visualized by reflection contrast microscopy and transmission and scanning electron microscopy
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DOI：
10.1097/00022744-200112000-00010
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发表时间：
2001-12-01
影响因子：
1.6
通讯作者：
van Dierendonck, JH
中科院分区：
文献类型：
--
作者：
Hoetelmans, RWM;Prins, FA;van Dierendonck, JH
作者最近发现，用丙酮、甲醇或多聚甲醛（PF）固定细胞单层后，再用甲醇（PF/甲醇）固定细胞单层后，Bcl-2和Bax蛋白的亚细胞免疫反应性发生了变化。在这里，作者通过反射对比显微镜和透射电子显微镜证明，与PF或戊二醛固定相比，丙酮或甲醇固定导致细胞内结构的完整性完全丧失。扫描电子显微镜显示，在丙酮或甲醇固定后，质膜的完整性保存较差。在甲醇之前用PF固定减少了对细胞内和质膜的损伤。此外，蛋白质印迹分析表明，Bcl-2和Bax蛋白在丙酮或甲醇固定过程中的损失，而甲醇透化前PF固定显着减少这种损失。对于可溶性或未知类型抗原的细胞内定位研究，作者不赞成使用丙酮和甲醇作为单一固定剂。
The authors recently showed variable subcellular immunoreactivity of the Bcl-2 and Bax proteins after fixation of cell monolayers with acetone, methanol, or paraformaldehyde (PF) followed by methanol (PF/methanol). Here, the authors demonstrate by reflection contrast microscopy and transmission electron microscopy that acetone or methanol fixation result in complete loss of integrity of intracellular structures in contrast with PF or glutaraldehyde fixation. Scanning electron microscopy revealed poor preservation of plasma membrane integrity after fixation in acetone or methanol. Fixation with PF before methanol reduced damage to intracellular and plasma membranes. In addition, Western blot analysis demonstrated loss of Bcl-2 and Bax protein during acetone or methanol fixation, whereas PF fixation before methanol permeabilization markedly reduced this loss. For studies on the intracellular localization of soluble or unknown types of antigen, the authors discourage the use of acetone and methanol as single fixatives.