Reversing the effects of formalin fixation with citraconic anhydride and heat: A universal antigen retrieval method
Reversing the effects of formalin fixation with citraconic anhydride and heat: A universal antigen retrieval method
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DOI：
10.1369/jhc.4c6466.2005
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发表时间：
2005-01-01
影响因子：
3.2
通讯作者：
Sugisaki, Y
中科院分区：
文献类型：
--
作者：
Namimatsu, S;Ghazizadeh, M;Sugisaki, Y
福尔马林是病理实验室常用的组织保存固定剂。这种固定剂的主要副作用是通过蛋白质交联隐藏组织抗原。为了实现恒定条件下免疫组织化学的通用抗原修复方法，我们开发了一种新方法，其中用柠康酸酐（一种可逆蛋白质交联剂）加加热来逆转福尔马林固定的影响。对来自不同器官的福尔马林固定、石蜡包埋的组织进行了多种抗原的免疫组织化学定位检查。将D组织切片放入含有0.05%柠康酸酐溶液（pH 7.4）的电厨房锅中，并将锅设置为98℃的“保温”温度模式45分钟。该模式允许在恒定温度下加热切片。然后将切片在缓冲溶液中洗涤，并使用自动染色机使用标记的链霉亲和素-生物素方法进行免疫染色。一般来说，福尔马林固定的组织表现出与新鲜冷冻组织相当的特异性免疫染色，并且比传统抗原修复方法显着增强。特别是，即使是难以检测的抗原，如 CD4、细胞周期蛋白 D1、颗粒酶 β、bcl-6、CD25 和 lambda 链，也显示出不同的免疫染色。不同类别的抗原，例如细胞标记物和受体，以及细胞质和核蛋白，始终产生增强的反应。该方法提供了有效的抗原修复，可在优化的条件下成功地对多种抗原进行免疫染色。它还允许病理实验室中福尔马林固定组织的免疫组织化学标准化，消除实验室间的结果差异，从而实现准确的临床和研究。
Formalin is a commonly used fixative for tissue preservation in pathology laboratories. A major adverse effect of this fixative is the concealing of tissue antigens by protein cross-linking. To achieve a universal antigen retrieval method for immunohistochemistry under a constant condition, we developed a new method in which the effects of formalin fixation were reversed with citraconic anhydride (a reversible protein cross-linking agent) plus heating. Formalin-fixed, paraffin-embedded tissues from various organs were examined for immunohistochemical localization of a wide variety of antigens. D tissue sections were placed in an electric kitchen pot containing 0.05% citraconic anhydride solution, pH 7.4, and the pot was set at "keep warm" temperature mode of 98C for 45 min. This mode allowed heating the sections at a constant temperature. The sections were then washed in buffer solution and immunostained using a labeled streptavidin-biotin method using an automated stainer. In general, formalin-fixed tissues demonstrated specific immunostainings comparable to that in fresh frozen tissues and significantly more enhanced than after conventional antigen retrieval methods. In particular, even difficult-to-detect antigens such as CD4, cyclin D1, granzyme beta, bcl-6, CD25, and lambda chain revealed distinct immunostainings. Different classes of antigens such as cellular markers and receptors, as well as cytoplasmic and nuclear proteins, consistently produced enhanced reactions. This method provides efficient antigen retrieval for successful immunostaining of a wide variety of antigens under an optimized condition. It also allows standardization of immunohistochemistry for formalin-fixed tissues in pathology laboratories, eliminating inter-laboratory discrepancies in results for accurate clinical and research studies.