Publication: Advantages and limitations of 3,3',5,5'-tetramethylbenzidine for immunohistochemical staining
Authors
Yu Chao ; Liu Xiao ; Zhao Peiyuan ; Sun Zhidong ; Song Yurong ; Cao Yuan ; Cheng Ming
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Publisher
Universidad de Murcia, Departamento de Histología e Histopatología
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DOI
https://doi.org/ 10.14670/HH-18-888
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info:eu-repo/semantics/article
Description
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
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Citation
Histology and Histopathology, volúmen 40, nº 10 (2025) 1667-1674
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