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.
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