TECHNIQUES TO OPTIMIZE POSTEMBEDDING SINGLE AND DOUBLE STAINING FOR AMINO-ACID NEUROTRANSMITTERS
TECHNIQUES TO OPTIMIZE POSTEMBEDDING SINGLE AND DOUBLE STAINING FOR AMINO-ACID NEUROTRANSMITTERS
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DOI：
10.1177/40.7.1376741
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发表时间：
1992-07-01
影响因子：
3.2
通讯作者：
RUSTIONI, A
中科院分区：
文献类型：
--
作者：
PHEND, KD;WEINBERG, RJ;RUSTIONI, A
我们报告了一些用于谷氨酸、天冬氨酸和伽马氨基丁酸的包埋后电子显微镜单染和双染的技术改进。在固定剂中加入2.5%的戊二醛，并通过最小化塑料聚合时间和切割与反应的间隔，可获得最佳的效果。提供了外源性谷氨酸、天冬氨酸和γ-氨基丁酸阻止其免疫染色的能力的定量文件。谷氨酸和天冬氨酸抗血清与天冬氨酸和天冬氨酸抗血清预先孵育后，谷氨酸和天冬氨酸抗血清的染色强度增加。对同一物种的抗血清进行双重染色时，首先要阻断第一抗血清的抗原性；用热的多聚甲醛烟雾进行双重染色效果最佳。通过使用洗涤剂而不是蚀刻，这些方法允许同时可视化示踪剂来识别神经解剖路径。
We report a number of technical refinements for single and double staining with post-embedding electron microscopy for glutamate, aspartate, and gamma-aminobutyric acid. Best results were obtained with 2.5% glutaraldehyde in the fixative and by minimizing the duration of plastic polymerization and the interval between cutting and reacting. Quantitative documentation of the ability of exogenous glutamate, aspartate, and gamma-aminobutyric acid to block their immune staining is provided. Increased intensity of staining with the glutamate and aspartate antisera resulted from preincubation of glutamate antiserum with aspartate and aspartate antiserum with glutamate. To perform double staining with antisera raised in the same species, it was necessary to block antigenicity of the first antiserum; best results were obtained with hot paraformaldehyde fumes. By using a detergent instead of etching, these methods permitted the simultaneous visualization of tracers to identify neuroanatomic pathways.