Ino Hidetoshi
Department of Neurobiology (C1), Graduate School of Medicine, Chiba University, Chiba, Japan.
J Histochem Cytochem. 2003 Aug;51(8):995-1003. doi: 10.1177/002215540305100803.
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.
在对存档的福尔马林固定、石蜡包埋组织切片进行成功的免疫组织化学（IHC）检测时，抗原修复（AR）常常是必需的。尽管通过加热进行抗原修复最为常用，但大多数现有方法仅适用于石蜡包埋切片。本文介绍了一种简单的替代抗原修复方法，该方法可用于醛固定冷冻切片。在多聚甲醛中固定后，将组织块在修复溶液中加热，然后用干冰冷冻。加热的最佳温度为90℃及以上，最佳修复溶液为蒸馏水和10 mM柠檬酸钠（pH 6.0）。用低温恒温器切片并将其置于涂有聚-L-赖氨酸的载玻片上。切片干燥后，进行常规免疫组织化学检测。或者，也可使用游离漂浮切片。该方法不仅大大增强了多种抗原的免疫反应性，尤其是核蛋白的免疫反应性，而且在某些情况下还有效降低了背景染色。我使用小鼠脑和大鼠睾丸切片检测了14种抗体的染色情况。加热过程对5种抗体至关重要，对7种抗体提高了免疫反应性，对2种抗体没有影响。
