A VERSATILE MEANS OF INTRACELLULAR LABELING - INJECTION OF BIOCYTIN AND ITS DETECTION WITH AVIDIN CONJUGATES
A VERSATILE MEANS OF INTRACELLULAR LABELING - INJECTION OF BIOCYTIN AND ITS DETECTION WITH AVIDIN CONJUGATES
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DOI：
10.1016/0165-0270(88)90114-8
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发表时间：
1988-08-01
影响因子：
3
通讯作者：
ARMSTRONG, WE
中科院分区：
文献类型：
--
作者：
HORIKAWA, K;ARMSTRONG, WE
生物胞素是一种低分子量的生物素-赖氨酸复合物，约65%的生物素内含有生物素，其保留了对抗生物素蛋白的高亲和力。由于后一种分子已经与几种组织化学标记物缀合，因此研究了生物胞素作为细胞内标记物的用途。用溶解在0.5M KCl和0.05M Tris缓冲液（pH 7-7.6）中的4-6%生物胞素的溶液填充电极。在视上核的rt视上神经垂体系统的外植体制备细胞内记录神经元，并注射超极化或去极化电流1-20分钟。在不同的恢复时间后，将外植体在10%福尔马林或4%多聚甲醛中固定过夜，在振动切片机上切片，并与抗生物素蛋白-生物素复合物（ABC）或已与荧光素、罗丹明、德克萨斯红或辣根过氧化物酶缀合并含有1% Triton-X 100的抗生物素蛋白孵育。使用每种标记回收了高百分比的注射神经元，并取得了大致相同的成功。可以使用负或正电流注入，几乎没有电极堵塞。荧光标记物的定性类似于荧光黄，而与辣根过氧化物酶或与ABC偶联的抗生物素蛋白标记的定性类似于直接用辣根过氧化物酶填充神经元。这种技术的优点是易于注射的biocytin和多功能性，让研究人员选择之间的发光和吸光图像。
Biocytin is a biotin-lysine complex of low molecular weight containing about 65% within biotin, which retains a high affinity for avidin. Since the latter molecule has been conjugated to several histochemical markers, the use of biocytin as an intracellular marker was investigated. Electrodes were filled with a solution of 4-6% biocytin dissolved in 0.5 M KCl and 0.05 M Tris buffer, pH 7-7.6. Neurons were recorded intracellularly in the supraoptic nucleus of an explant preparation of the rt supraoptico-neurohypophysial system and injected for 1-20 min with either hyperpolarizing or depolarizing current. Following variable recovery times, the explants were fixed in either 10% formalin or 4% paraformaldehyde overnight, sectioned on a vibratome, and incubated with the avidin-biotin complex (ABC) or avidin which had been conjugated to fluorescein, rhodamine, Texas Red or horseradish peroxidase and containing 1% Triton-X 100. A high percentage of injected neurons were recovered using each of the labels with about equal success. Both negative or positive current injection could be used with little electrode clogging. Labeling with fluorescent conjugates was qualitatively similar to that of Lucifer Yellow, whereas labeling with avidin coupled to horseradish peroxidase or with ABC was qualitatively similar to filling neurons directly with horseradish peroxidase. The advantages of this technique are the ease of injection of biocytin and the versatility in allowing the investigator to choose among light-emitting and light-absorbing images.