Mihelic-Rapp M, Giebel W
Department of Otorhinolaryngology, University of Tübingen, Germany.
Eur Arch Otorhinolaryngol. 1996;253(7):411-6. doi: 10.1007/BF00168493.
A new method was developed for frozen section detection of antigens that natively occur in the cochlear peri- and endolymph. A combination of immuno-histochemistry and immunoblot assay enabled topological and quantitative detection of small and hydrophilic molecules (such as the aminoglycoside antibiotics) in frozen sections of the inner ear compartments (scala tympani, scala vestibuli and cochlear duct). A selective localization is possible in the peri- and endolymphatic region of each coil of the cochlea. During sectioning of the cochlea, a small piece of a nitrocellulose membrane is placed to the surface of the intersection and briefly warmed. The sections are cut, simultaneously attached to a nitrocellulose membrane on which the aminoglycoside antibiotics remain adsorbed without any fixation procedure. Using this method, immunoincubation to detect gentamicin was performed in a way usually done in western blot analysis. Results with two different enzyme reactions with the enzyme conjugated to a second antibody (i.e., dye as substrate and the chemiluminescence detection system) are presented and compared. This histoimmunoblot assay provides a general non-radioactive and sensitive immunohistochemical tool for the localization of compounds occurring in extracellular body fluid compartments. For inner ear research this method now enables the investigation of the penetration and distribution of therapeutics in peri- and endolymphatic sites and can even be applied to separately quantifying concentrations of a substance in different coils of the same cochlear section.
开发了一种新方法，用于对耳蜗外淋巴和内淋巴中天然存在的抗原进行冰冻切片检测。免疫组织化学和免疫印迹分析相结合，能够对内耳腔室（鼓阶、前庭阶和蜗管）的冰冻切片中的小分子和亲水分子（如氨基糖苷类抗生素）进行拓扑和定量检测。在耳蜗每个蜗圈的外淋巴和内淋巴区域实现选择性定位是可能的。在耳蜗切片过程中，将一小片硝酸纤维素膜置于切片表面并短暂加热。切片被切割后，同时附着在硝酸纤维素膜上，氨基糖苷类抗生素无需任何固定程序即可吸附在该膜上。使用这种方法，以通常用于蛋白质印迹分析的方式进行检测庆大霉素的免疫孵育。展示并比较了与二抗偶联的酶的两种不同酶反应的结果（即染料作为底物和化学发光检测系统）。这种组织免疫印迹分析提供了一种通用的非放射性且灵敏的免疫组织化学工具，用于定位细胞外体液隔室中存在的化合物。对于内耳研究，这种方法现在能够研究治疗药物在内淋巴和外淋巴部位的渗透和分布，甚至可以用于单独定量同一耳蜗切片不同蜗圈中一种物质的浓度。
