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首页> 外文期刊>The Korean Journal of Physiology & Pharmacology >Optimized Immunohistochemical Analysis of Cerebellar Purkinje Cells Using a Specific Biomarker, Calbindin D28k
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Optimized Immunohistochemical Analysis of Cerebellar Purkinje Cells Using a Specific Biomarker, Calbindin D28k

机译：使用特定生物标记物Calbindin D28k优化小脑Purkinje细胞的免疫组织化学分析

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Cerebellar Purkinje cells (PCs) play a crucial role in motor functions and their progressive degeneration is closely associated with spinocerebellar ataxias. Although immunohistochemical (IHC) analysis can provide a valuable tool for understanding the pathophysiology of PC disorders, the method validation of IHC analysis with cerebellar tissue specimens is unclear. Here we present an optimized and validated IHC method using antibodies to calbindin D28k, a specific PC marker in the cerebellum. To achieve the desired sensitivity, specificity, and reproducibility, we modified IHC analysis procedures for cerebellar tissues. We found that the sensitivity of staining varies depending on the commercial source of primary antibody. In addition, we showed that a biotin-free signal amplification method using a horseradish peroxidase polymer-conjugated secondary antibody increases both the sensitivity and specificity of ICH analysis. Furthermore, we demonstrated that dye filtration using a 0.22 μm filter eliminates or minimizes nonspecific staining while preserving the analytical sensitivity. These results suggest that our protocol can be adapted for future investigations aiming to understand the pathophysiology of cerebellar PC disorders and to evaluate the efficacy of therapeutic strategies for treating these diseases.
机译：小脑浦肯野细胞（PCs）在运动功能中起关键作用，其进行性变性与脊髓小脑共济失调密切相关。尽管免疫组化（IHC）分析可以为了解PC疾病的病理生理提供有价值的工具，但尚不清楚用小脑组织标本进行IHC分析的方法验证。在这里，我们提出了一种经过优化和验证的IHC方法，该方法使用针对小分子中特定PC标志物calbindin D28k的抗体。为了达到所需的灵敏度，特异性和可重复性，我们修改了小脑组织的IHC分析程序。我们发现染色的敏感性根据一抗的商业来源而变化。此外，我们表明使用辣根过氧化物酶聚合物偶联的二抗的无生物素信号放大方法可提高ICH分析的灵敏度和特异性。此外，我们证明了使用0.22μm过滤器进行染料过滤可以消除或最小化非特异性染色，同时保持分析灵敏度。这些结果表明，我们的方案可适用于将来的研究，旨在了解小脑PC疾病的病理生理学并评估治疗这些疾病的治疗策略的有效性。

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