Non-specific background in immunoglobulin G staining can be effectively eliminated using heating or a catalase inhibitor

IF 2.3 4区 生物学 Q4 CELL BIOLOGY
Kairan Yang , Ting Xu , Chengkang Lin , Zuisu Yang , Haiyan Lyu , Falei Yuan
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引用次数: 0

Abstract

Immunoglobulin G (IgG) staining is a widely used method for assessing blood-brain barrier (BBB) integrity. However, significant non-specific binding is often observed in many studies, which can interfere with the accurate interpretation of results. In this study, the horseradish peroxidase (HRP)-based polymer method and the streptavidin-biotin complex (SABC) method were used to perform IgG staining. The effects of hydrogen peroxide, heating, and a catalase inhibitor on reducing background staining were evaluated in brain sections from untreated mice and those subjected to middle cerebral artery occlusion (MCAO). The results showed that immunohistochemistry without hydrogen peroxide pretreatment still produced minimal background in paraffin-embedded sections. However, IgG staining with hydrogen peroxide pretreatment led to substantial background in vibratome sections. Compared to the SABC method, a mixture of the catalase inhibitor 3-amino-1,2,4-triazole and hydrogen peroxide reduced background staining by 35.4 % ± 5.7 % in the cortex of untreated mouse brains and by 36.9 % ± 1.8 % in the contralateral cortex of MCAO mice when using the polymer method. Additionally, heating at 75°C was sufficient to eliminate non-specific binding in brain sections from both untreated and MCAO mice. Hydrogen peroxide pretreatment alone was ineffective in removing background staining in brain sections from either untreated or MCAO mice. In summary, this study demonstrates that hydrogen peroxide pretreatment is effective in reducing background only when combined with a catalase inhibitor but is unnecessary when the tissue is heated. Heating is a simple and effective method for removing the IgG staining background when detecting BBB leakage.
免疫球蛋白G染色中的非特异性背景可以通过加热或过氧化氢酶抑制剂有效地消除
免疫球蛋白G (IgG)染色是一种广泛使用的评估血脑屏障(BBB)完整性的方法。然而,在许多研究中经常观察到显著的非特异性结合,这可能会干扰结果的准确解释。本研究采用辣根过氧化物酶(HRP)聚合物法和链亲和素-生物素复合物(SABC)法进行IgG染色。过氧化氢、加热和过氧化氢酶抑制剂对降低背景染色的影响在未治疗的小鼠和大脑中动脉闭塞(MCAO)小鼠的脑切片中进行了评估。结果表明,未经双氧水预处理的免疫组织化学方法在石蜡包埋切片中仍产生极小的背景。然而,过氧化氢预处理的IgG染色在振动体切片中导致大量背景。与SABC方法相比,过氧化氢酶抑制剂3-氨基-1,2,4-三唑和过氧化氢的混合物在使用聚合物方法时,未处理小鼠大脑皮层的背景染色减少35.4% % ± 5.7 %,在MCAO小鼠对侧皮层的背景染色减少36.9 % ± 1.8 %。此外,在75°C下加热足以消除未处理和MCAO小鼠脑切片中的非特异性结合。单独过氧化氢预处理对未处理或MCAO小鼠脑切片的背景染色均无效。总之,这项研究表明,过氧化氢预处理只有在与过氧化氢酶抑制剂联合使用时才能有效地降低背景,而当组织被加热时则没有必要。加热是检测血脑屏障渗漏时去除IgG染色背景的一种简单有效的方法。
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来源期刊
Acta histochemica
Acta histochemica 生物-细胞生物学
CiteScore
4.60
自引率
4.00%
发文量
107
审稿时长
23 days
期刊介绍: Acta histochemica, a journal of structural biochemistry of cells and tissues, publishes original research articles, short communications, reviews, letters to the editor, meeting reports and abstracts of meetings. The aim of the journal is to provide a forum for the cytochemical and histochemical research community in the life sciences, including cell biology, biotechnology, neurobiology, immunobiology, pathology, pharmacology, botany, zoology and environmental and toxicological research. The journal focuses on new developments in cytochemistry and histochemistry and their applications. Manuscripts reporting on studies of living cells and tissues are particularly welcome. Understanding the complexity of cells and tissues, i.e. their biocomplexity and biodiversity, is a major goal of the journal and reports on this topic are especially encouraged. Original research articles, short communications and reviews that report on new developments in cytochemistry and histochemistry are welcomed, especially when molecular biology is combined with the use of advanced microscopical techniques including image analysis and cytometry. Letters to the editor should comment or interpret previously published articles in the journal to trigger scientific discussions. Meeting reports are considered to be very important publications in the journal because they are excellent opportunities to present state-of-the-art overviews of fields in research where the developments are fast and hard to follow. Authors of meeting reports should consult the editors before writing a report. The editorial policy of the editors and the editorial board is rapid publication. Once a manuscript is received by one of the editors, an editorial decision about acceptance, revision or rejection will be taken within a month. It is the aim of the publishers to have a manuscript published within three months after the manuscript has been accepted
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