Carmichael-Lowe Amber, Fleming Brionne, Reddy Kreesan, Wiseman James, Yin Eden Paige, Turner Clinton P, Faull Richard L M, Curtis Maurice A, Dragunow Mike, Dieriks Birger Victor
Department of Anatomy and Medical Imaging, University of Auckland, Auckland, New Zealand.
Centre for Brain Research, University of Auckland, Auckland, New Zealand.
PLoS One. 2024 Dec 17;19(12):e0315183. doi: 10.1371/journal.pone.0315183. eCollection 2024.
Antigen retrieval is crucial for immunohistochemistry, particularly in formalin-fixed paraffin-embedded brain tissue, where fixation causes extensive crosslinking that masks epitopes. Heat Induced Epitope Retrieval (HIER) reverses these crosslinks, improving access to nuclear and aggregated proteins. We introduce Cyclic Heat-Induced Epitope Retrieval (CHIER), an advanced technique that builds on HIER by incorporating repeated cycles of heating and cooling. CHIER optimises antigen retrieval and significantly improves detection. CHIER is particularly effective for detecting chromatin-binding proteins, such as SMARCC2, which are difficult to label using conventional IHC methods. Using CHIER on formalin-fixed paraffin-embedded human brain sections, we achieved robust detection of SMARCC2 in both the nucleus and cytoplasm. CHIER also enhanced the visualisation of large SMARCC2+ cytoplasmic bodies, termed cytobodies, which are increased in Parkinson's Disease (PD). Our findings suggest that SMARCC2 may translocate from the nucleus to the cytoplasm in PD, potentially implicating SMARCC2 aggregation in the disease's pathology. Furthermore, CHIER does not negatively impact the antigenicity of other antibodies, supporting its use for multiplex fluorescent immunohistochemistry and super-resolution imaging. These results highlight CHIER's potential for improving the detection of chromatin-binding and aggregated proteins in neurodegenerative disease research, offering new insights into SMARCC2's role in Parkinson's Disease.
抗原修复对于免疫组织化学至关重要，尤其是在福尔马林固定石蜡包埋的脑组织中，固定会导致广泛的交联，从而掩盖表位。热诱导表位修复（HIER）可逆转这些交联，改善对核蛋白和聚集蛋白的检测。我们引入了循环热诱导表位修复（CHIER），这是一种先进技术，它在HIER的基础上，通过纳入反复的加热和冷却循环来构建。CHIER优化了抗原修复并显著提高了检测效果。CHIER对于检测染色质结合蛋白（如SMARCC2）特别有效，而使用传统免疫组化方法难以标记这些蛋白。在福尔马林固定石蜡包埋的人脑切片上使用CHIER，我们在细胞核和细胞质中均实现了对SMARCC2的可靠检测。CHIER还增强了称为细胞体的大型SMARCC2 +细胞质体的可视化，这些细胞体在帕金森病（PD）中会增加。我们的研究结果表明，在PD中SMARCC2可能从细胞核转移到细胞质，这可能暗示SMARCC2聚集与该疾病的病理有关。此外，CHIER不会对其他抗体的抗原性产生负面影响，支持其用于多重荧光免疫组化和超分辨率成像。这些结果突出了CHIER在神经退行性疾病研究中改善染色质结合和聚集蛋白检测的潜力，为SMARCC2在帕金森病中的作用提供了新的见解。
