STABILIZATION OF THE TETRAMETHYLBENZIDINE (TMB) REACTION-PRODUCT - APPLICATION FOR RETROGRADE AND ANTEROGRADE TRACING, AND COMBINATION WITH IMMUNOHISTOCHEMISTRY
STABILIZATION OF THE TETRAMETHYLBENZIDINE (TMB) REACTION-PRODUCT - APPLICATION FOR RETROGRADE AND ANTEROGRADE TRACING, AND COMBINATION WITH IMMUNOHISTOCHEMISTRY
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DOI：
10.1177/32.11.6548485
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发表时间：
1984-01-01
影响因子：
3.2
通讯作者：
WAINER, BH
中科院分区：
文献类型：
--
作者：
RYE, DB;SAPER, CB;WAINER, BH
四甲基联苯胺（TMB）作为辣根过氧化物酶（HRP）组织化学的底物比其他显色剂更敏感。然而，其在水溶液和乙醇中的不稳定性限制了其应用。报道了通过在二氨基联苯胺（DAB）/钴（Co 2+）/H2 O2组合中孵育来稳定TMB的方法。稳定的TMB产品不受长期暴露于乙醇、中性缓冲液和随后的免疫组织化学染色程序的影响。推荐一个程序，以最佳的TMB稳定，提供了一个灵敏度，证明逆行标记胞体可比标准TMB组织化学。反应产物的物理特性使其适合与未标记抗体、过氧化物酶-抗过氧化物酶（PAP）免疫组织化学染色程序结合。这是通过用抗胆碱乙酰转移酶的单克隆抗体对大鼠基底前脑中的逆行标记神经元进行染色而建立的。由于稳定的TMB产物表现出优于基于DAB的反应产物（DAB-Co）的Co2+强化上级灵敏度，因此其提供了优于先前描述的组合方法的明显优势。
Tetramethylbenzidine (TMB) as a substrate for horseradish peroxidase (HRP) histochemistry is more sensitive than other chromogens. Its instability in aqueous solutions and ethanol, however, has limited its application. A method is reported for stabilizing TMB by incubation in combinations of diaminobenzidine (DAB)/cobalt (Co2+)/H2O2. The stabilized TMB product was unaffected by long-term exposures to ethanol, neutral buffers and subsequent immunohistochemical staining procedures. A procedure is recommended for optimal stabilization of TMB that affords a sensitivity for demonstrating retrogradely labeled perikarya comparable to standard TMB histochemistry. The physical characteristics of the reaction product make it suitable for combination with the unlabeled antibody, peroxidase-antiperoxidase (PAP) immunohistochemical staining procedure. This was established by staining retrogradely labeled neurons in the rat basal forebrain with a monoclonal antibody against choline acetyltransferase. Because the stabilized TMB product exhibited a superior sensitivity over Co2+ intensification of the DAB-based reaction product (DAB-Co), it offers a distinct advantage over previously described combination procedures.