Ghosh Sumana, Lewis Mark B, Walters Bradley J
Department of Neurobiology and Anatomical Sciences University of Mississippi Medical Center Jackson Mississippi USA.
Department Otolaryngology-Head and Neck Surgery University of Mississippi Medical Center Jackson Mississippi USA.
Laryngoscope Investig Otolaryngol. 2020 Aug 26;5(5):919-927. doi: 10.1002/lio2.449. eCollection 2020 Oct.
The pervasiveness of hearing loss and the development of new potential therapeutic approaches have led to increased animal studies of the inner ear. However, translational relevance of such studies depends upon verification of protein localization data in human samples. Cadavers used for anatomical education provide a potential research resource, but are limiting due to difficulties in accessing sensory tissues from the dense temporal bones. This study seeks to reduce the often months-long process of decalcification and improve immunofluorescent staining of human cadaveric temporal bones for research use.
Temporal bones were decalcified in either (a) hydrochloric acid-containing RDO solution for 2 days followed by 0.5 M ethylenediaminetetraacetic acid (EDTA) for 3 to 5 additional days, or (b) 0.5 M EDTA alone for 2 to 4 weeks. Image-iT FX signal enhancer (ISE) was used to improve immunofluorescent signal-to-noise ratios.
The data indicate that both methods speed decalcification and allow for immunolabeling of the extranuclear proteins neurofilament (heavy chain), myosin VIIa, oncomodulin and prestin. However, RDO decalcification was more likely to alter structural morphology of sensory tissues and hindered effective labeling of the nuclear proteins SRY-box transcription factor 2 and GATA binding protein 3.
Although both approaches allow for rapid decalcification, EDTA appears superior to RDO for preserving cytoarchitecture and immunogenicity.
NA.
听力损失的普遍性以及新的潜在治疗方法的发展，使得内耳的动物研究有所增加。然而，此类研究的转化相关性取决于对人类样本中蛋白质定位数据的验证。用于解剖学教学的尸体提供了一种潜在的研究资源，但由于难以从致密的颞骨中获取感觉组织，其应用受到限制。本研究旨在缩短通常长达数月的脱钙过程，并改善用于研究的人类尸体颞骨的免疫荧光染色。
颞骨在以下两种方法中进行脱钙：(a) 在含盐酸的RDO溶液中脱钙2天，然后在0.5 M乙二胺四乙酸（EDTA）中再脱钙3至5天；或(b) 仅用0.5 M EDTA脱钙2至4周。使用Image-iT FX信号增强剂（ISE）来提高免疫荧光信噪比。
数据表明，两种方法都能加快脱钙速度，并允许对核外蛋白神经丝（重链）、肌球蛋白VIIa、癌调蛋白和预应力蛋白进行免疫标记。然而，RDO脱钙更有可能改变感觉组织的结构形态，并阻碍对核蛋白SRY盒转录因子2和GATA结合蛋白3的有效标记。
虽然两种方法都能实现快速脱钙，但在保存细胞结构和免疫原性方面，EDTA似乎优于RDO。
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