Advantages and limitations of 3,3',5,5'-tetramethylbenzidine for immunohistochemical staining.

IF 2.5 4区 生物学 Q3 CELL BIOLOGY
Chao Yu, Xiao Liu, Peiyuan Zhao, Zhidong Sun, Yurong Song, Yuan Cao, Ming Cheng
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引用次数: 0

Abstract

In this study, two chromogenic systems, horseradish peroxidase (HRP)-3,3'-diaminobenzidine (DAB) and HRP-3,3',5,5'-tetramethylbenzidine (TMB), were used to perform single-color and double-color immunohistochemical staining (sIHC and dIHC, respectively) on multiple antigens in four distinct tissue types. The chromogenic results of the HRP-TMB system exhibited a vibrant blue-green color, and the tissue localization and signal intensity were consistent with those of the HRP-DAB system. In addition, it demonstrated clear differentiation from the hematoxylin-stained nucleus, endogenous melanin, and brown chromogenic results of HRP-DAB. TMB staining in tissues that contain high endogenous pigment levels eliminates the need for melanin bleaching, thereby facilitating direct observation and potentially improving the detection speed and interpretation. TMB can also be used in combination with DAB for dIHC, thus allowing detection of the two markers on a single slide. However, the TMB staining results are not stable over the long term and require image storage using slide scanners, thereby limiting its application. Additionally, in dIHC, overlapping signals of the first marker may obscure the second marker, potentially leading to bias or false negatives. Therefore, careful consideration is required when designing dIHC detection systems. Based on the above, we propose that TMB is a valuable supplement to DAB for immunohistochemical staining and deserves further promotion and utilization. However, additional research is needed to improve the composition of TMB chromogenic reagents, prolong the longevity of staining results, and overcome current limitations.

本研究采用辣根过氧化物酶(HRP)-3,3'-二氨基联苯胺(DAB)和 HRP-3,3',5,5'-四甲基联苯胺(TMB)两种发色系统,分别对四种不同组织类型的多种抗原进行单色和双色免疫组化染色(sIHC 和 dIHC)。HRP-TMB 系统的显色结果呈鲜艳的蓝绿色,组织定位和信号强度与 HRP-DAB 系统一致。此外,它还与苏木精染色的细胞核、内源性黑色素和 HRP-DAB 的棕色显色结果有明显区别。在内源性色素含量较高的组织中进行 TMB 染色,无需对黑色素进行漂白,从而方便了直接观察,并有可能提高检测速度和判读能力。TMB 还可与 DAB 结合用于 dIHC,从而在一张载玻片上检测两种标记物。不过,TMB 染色结果并不长期稳定,需要使用载玻片扫描仪进行图像存储,因此限制了其应用。此外,在 dIHC 中,第一个标记物的重叠信号可能会遮盖第二个标记物,从而可能导致偏差或假阴性。因此,在设计 dIHC 检测系统时需要慎重考虑。综上所述,我们认为 TMB 是 DAB 用于免疫组化染色的重要补充,值得进一步推广和使用。然而,如何改进 TMB 发色试剂的成分、延长染色结果的寿命并克服目前的局限性,还需要进一步的研究。
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来源期刊
Histology and histopathology
Histology and histopathology 生物-病理学
CiteScore
3.90
自引率
0.00%
发文量
232
审稿时长
2 months
期刊介绍: HISTOLOGY AND HISTOPATHOLOGY is a peer-reviewed international journal, the purpose of which is to publish original and review articles in all fields of the microscopical morphology, cell biology and tissue engineering; high quality is the overall consideration. Its format is the standard international size of 21 x 27.7 cm. One volume is published every year (more than 1,300 pages, approximately 90 original works and 40 reviews). Each volume consists of 12 numbers published monthly online. The printed version of the journal includes 4 books every year; each of them compiles 3 numbers previously published online.
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