{"title":"Advantages and limitations of 3,3',5,5'-tetramethylbenzidine for immunohistochemical staining.","authors":"Chao Yu, Xiao Liu, Peiyuan Zhao, Zhidong Sun, Yurong Song, Yuan Cao, Ming Cheng","doi":"10.14670/HH-18-888","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":13164,"journal":{"name":"Histology and histopathology","volume":" ","pages":"18888"},"PeriodicalIF":2.5000,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Histology and histopathology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.14670/HH-18-888","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"CELL BIOLOGY","Score":null,"Total":0}
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.
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.