乙醛固定剂使环AMP与脑组织成分交联的证据:在组织化学过程中的潜在效用。

T W Rall, R A Lehne
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引用次数: 0

摘要

通过免疫组织化学方法从未固定的脑组织切片中回收的环AMP的量很小(小于2 pmol /mg蛋白质),尽管在冷冻和切片前积累的量有很大的变化,但仍是恒定的。然而,用戊二醛或甲醛在缓冲蔗糖溶液中处理脑切片,在0度下处理一到两个小时,通过过滤处理组织的匀浆来判断,累积的总环AMP的不溶性高达60%。免疫反应性环AMP通过温稀酸萃取从过滤器中回收。在较宽的初始组织水平范围内,过滤器结合环AMP的分数相对恒定。当pH维持在8.5以上时,不溶性环AMP在甲醛处理的切片匀浆中的持久性最大;在0度下加热两小时后,约有40%的这一组分丢失。用甲醛和放射性核苷或腺嘌呤、鸟嘌呤和胞嘧啶的核苷酸衍生物孵育组织匀浆,导致不溶性放射性的时间依赖性外观;缺乏氨基功能的化合物是无活性的。纯蛋白质,包括聚赖氨酸,也与甲醛和3h -环AMP反应,产生抗木炭吸附的放射性。用紫外光谱法测定了环AMP或腺苷、甲醛和烷基胺的反应。初步得出结论,在0℃时,甲醛能够在伯烷基胺和嘌呤核苷或核苷酸的n6氨基功能之间迅速产生亚甲基桥接加合物。将这些结果应用于环AMP细胞定位的免疫组织化学方法的发展,将需要产生对n6位置衍生的环AMP具有高反应性的抗体制剂。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Evidence for cross-linking of cyclic AMP to constituents of brain tissue by aldehyde fixatives: potential utility in histochemical procedures.

The amount of cyclic AMP recovered from unfixed tissue sections of brain carried through immunohistochemical procedures was found to be small (less than 2 pmoles/mg protein) and constant despite wide variations in the amount accumulated prior to freezing and sectioning. However, treatment of brain slices with glutaraldehyde or formaldehyde in buffered sucrose solutions for one to two hours at 0 degrees rendered insoluble as much as 60% of the total accumulated cyclic AMP as judged by filtration of homogenates of treated tissue. Immunoreactive cyclic AMP was recovered from filters by extraction with warm, dilute acid. The fraction of filter-bound cyclic AMP was relatively constant over a wide range of initial tissue levels. The persistence of insoluble cyclic AMP was greatest in homogenates of slices treated with formaldehyde when maintained above pH 8.5; about 40% of this fraction was lost after two hours at 0 degrees. Incubation of tissue homogenates with formaldehyde and radioactive nucleoside or nucleotide derivatives of adenine, guanine, and cytosine resulted in a time-dependent appearance of insoluble radioactivity; compounds lacking an amino function were inactive. Pure proteins, including polylysine, also reacted with formaldehyde and 3H-cyclic AMP to produce radioactivity resistant to adsorption by charcoal. The reaction between cyclic AMP or adenosine, formaldehyde, and alkyl amines was examined using UV spectrometry. It is tentatively concluded that at 0 degrees formaldehyde is capable of rapidly producing a methylene-bridged adduct between primary alkylamines and the N6-amino function of purine nucleosides or nucleotides. Application of these results to the development of immunohistochemical procedures for the cellular localization of cyclic AMP will require the generation of antibody preparations with high reactivity for cyclic AMP derivatized at the N6-position.

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