Section of Otolaryngology-Head and Neck Surgery, Department of Surgery, University of Chicago, Chicago, Illinois 60637-1447, USA.
Laryngoscope. 2011 Jul;121(7):1525-31. doi: 10.1002/lary.21772. Epub 2011 Jun 6.
OBJECTIVES/HYPOTHESIS: Immunofluorescence staining methods have been developed to study the distribution of macromolecules in archival formalin-fixed celloidin-embedded human temporal bone tissues. The aim of this study was to investigate the feasibility of utilizing this approach to evaluate the codistribution of more than one molecule of interest in a single tissue section.
Retrospective study of proteoglycan codistribution in archival human temporal bone tissues.
The chondroitin sulfate and keratan sulfate proteoglycans were selected for evaluating this methodology. Human tissues with known proteoglycan staining patterns were studied as controls. Thirty-one formalin-fixed celloidin-embedded archival human temporal bones were evaluated, and the observations in 11 specimens are described. A dual immunofluorescence staining method was developed using primary antibodies of differing isotypes and secondary antibodies labeled with fluorophores having nonoverlapping emission characteristics.
The specificity of the dual immunofluorescence technique for chondroitin sulfate and keratan sulfate proteoglycans was demonstrated in control tissues and confirmed through inhibition studies. The normal human tectorial membrane exhibited intense chondroitin sulfate staining. Cochlear and vestibular hair cells exhibited predominantly keratan sulfate staining. Keratan sulfate staining predominated in spiral ganglion cell bodies and fibers. Alterations in the normal distribution pattern of proteoglycans were observed in cases of presbycusis and otosclerosis.
The dual immunofluorescence staining methodology can be used to study archival formalin-fixed celloidin-embedded human temporal bone tissues. This technique may be applied to the evaluation of other molecules in archival human temporal bone tissues and lead to improvement in our understanding of the function of these molecules and their role in disease processes.
目的/假设：已经开发出免疫荧光染色方法来研究大分子在存档福尔马林固定细胞素包埋的人颞骨组织中的分布。本研究的目的是研究利用这种方法评估单个组织切片中一种以上感兴趣分子共分布的可行性。
对存档的人颞骨组织中蛋白聚糖共分布的回顾性研究。
选择硫酸软骨素和硫酸角质素蛋白聚糖来评估这种方法。研究了具有已知蛋白聚糖染色模式的人组织作为对照。评估了 31 个福尔马林固定细胞素包埋的存档人颞骨，并描述了 11 个标本的观察结果。使用不同同型的初级抗体和用具有不重叠发射特性的荧光标记的二级抗体开发了双重免疫荧光染色方法。
在对照组织中证明了双重免疫荧光技术对硫酸软骨素和硫酸角质素蛋白聚糖的特异性，并通过抑制研究得到证实。正常人类的听膜显示出强烈的硫酸软骨素染色。耳蜗和前庭毛细胞主要显示出硫酸角质素染色。螺旋神经节细胞体和纤维中硫酸角质素染色为主。在老年性聋和耳硬化症病例中观察到蛋白聚糖正常分布模式的改变。
双重免疫荧光染色方法可用于研究存档福尔马林固定细胞素包埋的人颞骨组织。该技术可应用于评估存档人颞骨组织中的其他分子，并有助于提高我们对这些分子功能及其在疾病过程中作用的理解。
