TISSUE FIXATION WITH DIIMIDOESTERS AS AN ALTERNATIVE TO ALDEHYDES I. COMPARISON OF CROSS-LINKING AND ULTRASTRUCTURE OBTAINED WITH DIMETHYLSUBERIMIDATE AND GLUTARALDEHYDE
TISSUE FIXATION WITH DIIMIDOESTERS AS AN ALTERNATIVE TO ALDEHYDES I. COMPARISON OF CROSS-LINKING AND ULTRASTRUCTURE OBTAINED WITH DIMETHYLSUBERIMIDATE AND GLUTARALDEHYDE
复制标题
使用二酰亚胺酯作为醛的替代品进行组织固定 I. 使用辛二酰亚胺二甲酯和戊二醛获得的交联和超微结构的比较
DOI：
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发表时间：
1974
影响因子：
3.2
通讯作者：
A. Hand
中科院分区：
文献类型：
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作者：
J. Hassell;A. Hand
二亚胺酯是一种双功能试剂，它与赖氨酸的ε-氨基反应，在极小程度上改变蛋白质的天然性质的情况下，形成分子间的交联键。为了确定二亚胺酯固定组织的最佳条件，基于分子间交联形成的蛋白质聚集体是不溶于水的假设，建立了一种交联法。用该方法测定新鲜肝脏中不溶性蛋白质的比例为16%，而与二亚胺酯(DMS)(16~20 mg/ml，0.02M Ca~(++)和0.15M Tris-HCl，pH 9.5)的最佳交联剂条件使92.1%的蛋白质不溶于固定液，只允许3.3%的蛋白质扩散到固定液中。较小的二亚胺酯，二甲基二乙醇酯和二甲基丙二酰亚胺，分别使74%和16%的蛋白质不溶。用不同的醛固定产生了从74%到90%以上的不溶性组分。在超微结构上，DMS固定组和戊二醛固定组的肝细胞形态相似，不同之处在于前者使高尔基球囊和滑面内质网扩张，线粒体基质电子密度增加。此外，戊二醛和DMS固定的肝脏的出现更容易与交联度相关，而不是与固定液本身的pH值相关。这项研究的结果表明，DMS是一种潜在的有用的光学和电子显微镜固定剂。
Diimidoesters are bifunctional reagents which react with the ε-amino group of lysine, forming intermolecular cross-links with minimal alteration of the native properties of proteins. To determine the optimal conditions for tissue fixation with diimidoesters, a cross-linking assay was developed based on the assumption that protein aggregates formed by intermolecular cross-links would be water-insoluble. With this assay method, the proportion of insoluble protein in fresh liver was 16%, while optimal conditions for cross-linking with the diimidoester, dimethylsuberimidate (DMS) (16-20 mg/ml, 0.02 M Ca++ and 0.15 M Tris-HCl, pH 9.5), rendered 92.1% insoluble and permitted only 3.3% of the protein to diffuse into the fixative solution. The smaller diimidoesters, dimethyladipimate and dimethylmalonimidate, rendered 74 and 16% of the protein insoluble, respectively. Fixation with various aldehydes produced insoluble fractions varying from 74% to over 90%. Ultrastructurally, hepatocytes of DMS-fixed liver and glutaraldehyde-fixed liver were similar except that with the former fixative the Golgi saccules and smooth endoplasmic reticulum were dilated and mitochondrial matrices exhibited an increased electron density. Furthermore, the appearance of glutaraldehyde- and DMS-fixed liver was more readily correlated with the degree of cross-linking than with the pH of the fixative solution per se. The results of this study indicate that DMS is a potentially useful fixative for light and electron microscopy.