Antigen retrieval by heating en bloc for pre-fixed frozen material
Antigen retrieval by heating en bloc for pre-fixed frozen material
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DOI：
10.1177/002215540305100803
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发表时间：
2003-08-01
影响因子：
3.2
通讯作者：
Ino, H
中科院分区：
文献类型：
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作者：
Ino, H
抗原检索（AR）是经常需要成功的免疫组织化学（IHC）档案福尔马林固定，石蜡包埋组织切片。虽然通过加热的AR是最常用的，但大多数现有的方法只适用于石蜡包埋的切片。本文描述了一种简单的AR替代方法，可用于醛固定冷冻切片。在多聚甲醛中固定后，组织块在回收液中加热，然后用干冰冷冻。最佳加热温度为90℃及以上，最佳提取液为蒸馏水和10 mM柠檬酸钠，pH为6.0。切片用低温恒温器切割，并安装在聚(L)赖氨酸涂层玻璃载玻片上。切片干燥后，进行常规免疫组化。或者，使用自由浮动部分。这种方法不仅大大提高了对多种抗原的免疫反应性，特别是对核蛋白的免疫反应性，而且在某些情况下有效地降低了背景染色。我用小鼠大脑和大鼠睾丸的切片检查了14种抗体的染色。加热过程对5种抗体是必需的，对7种抗体的免疫反应性有所提高，对2种抗体的免疫反应性没有变化。
Antigen retrieval (AR) is frequently required for successful immunohistochemistry (IHC) in archival formalin-fixed, paraffin-embedded tissue sections. Although AR by heating is most generally used, the majority of existing methods are useful only for paraffin-embedded sections. This article describes a simple alternative method for AR that can be used for aldehyde-fixed frozen sections. After fixation in paraformaldehyde, tissue blocks were heated in retrieval solutions and then frozen with dry ice. The optimal temperatures for heating were 90C and above, and the optimal retrieval solutions were distilled water and 10 mM sodium citrate, pH 6.0. Sections were cut with a cryostat and mounted on poly-(L)-lysine-coated glass slides. After the sections dried, routine IHC was performed. Alternatively, free-floating sections were used. This method not only greatly enhanced the immunoreactivity for a wide range of antigens, especially for nuclear proteins, but also effectively lowered the background staining in some cases. I examined the staining of 14 antibodies using sections of mouse brain and rat testis. The heating process was essential for five antibodies, improved immunoreactivity for seven antibodies, and provided no change for two antibodies.