Kaefer Samuel L, Shay Elizabeth O, Morrison Rachel A, Zhang Lujuan, Voytik-Harbin Sherry, Halum Stacey
Indiana University School of Medicine (IUSM) Indianapolis Indiana USA.
Department of Otolaryngology-Head and Neck Surgery IUSM Indianapolis Indiana USA.
Laryngoscope Investig Otolaryngol. 2025 Jan 7;10(1):e70020. doi: 10.1002/lio2.70020. eCollection 2025 Feb.
The current gold standard for immunofluorescent (IF) visualization of neuromuscular junctions (NMJs) in muscle utilizes frozen tissue sections with fluorescent conjugated antibodies to demarcate neurons and IF alpha-bungarotoxin (α-BTX) to demarcate motor endplates. Frozen tissue sectioning comes with inherent inescapable limitations, including cryosectioning artifact and limited sample shelf-life. However, a parallel approach to identify NMJs in paraffin-embedded tissue sections has not been previously described.
Yucatan minipig thyroarytenoid (TA) muscle was harvested and prepared as 5-μm thick paraffin-embedded tissue sections. A variety of antibodies at various concentrations were selected to target nicotinic acetylcholine receptors, synaptic vesicles, and neurons.
Neurofilament (NEFL, Invitrogen, 1:500) and synaptic vesicle glycoprotein (SV2, DSHB, 1:230) bound and demarcated the neurons and synaptic vesicles, respectively. Following consistent visualization of nerve tissue, rabbit anti-nicotinic acetylcholine receptor alpha-1 subunit (CHRNα, Abcam, 1:500) was used to identify the acetylcholine receptors within motor endplates. Complete NMJ visualization was then achieved with an optimized protocol using primary antibodies to the neurofilament light chain, nerve synaptic vesicle glycoprotein 2, and the alpha 1 subunit of the nicotinic acetylcholine receptor. Slide imaging was performed with the Echo Revolve Microscope (40×).
Herein, we describe a new methodology to visualize NMJs within paraffin-embedded TA muscle sections. Our protocol avoids the known limitations associated with cryosectioned samples and introduces a new neurolaryngologic research tool that utilizes the advantageous ability of paraffin-embedded sectioning to preserve tissue morphology. In conjunction with standard cryosectioned methods, the described paraffin-embedded protocol serves to enhance histological analysis of NMJs.
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目前用于肌肉中神经肌肉接头（NMJ）免疫荧光（IF）可视化的金标准方法是利用冷冻组织切片，用荧光偶联抗体标记神经元，并用IFα-银环蛇毒素（α-BTX）标记运动终板。冷冻组织切片存在一些固有的、不可避免的局限性，包括冷冻切片伪像和有限的样本保质期。然而，此前尚未描述过在石蜡包埋组织切片中识别NMJ的平行方法。
采集尤卡坦小型猪的甲杓肌（TA），制备成5μm厚的石蜡包埋组织切片。选择了各种不同浓度的抗体来靶向烟碱型乙酰胆碱受体、突触小泡和神经元。
神经丝（NEFL，Invitrogen，1:500）和突触小泡糖蛋白（SV2，DSHB，1:230）分别结合并标记了神经元和突触小泡。在神经组织得到一致的可视化后，使用兔抗烟碱型乙酰胆碱受体α-1亚基（CHRNα，Abcam，1:500）来识别运动终板内的乙酰胆碱受体。然后通过使用针对神经丝轻链、神经突触小泡糖蛋白2和烟碱型乙酰胆碱受体α1亚基的一抗的优化方案，实现了完整的NMJ可视化。使用Echo Revolve显微镜（40×）进行载玻片成像。
在此，我们描述了一种在石蜡包埋的TA肌肉切片中可视化NMJ的新方法。我们的方案避免了与冷冻切片样本相关的已知局限性，并引入了一种新的神经喉科学研究工具，该工具利用石蜡包埋切片保留组织形态的优势能力。与标准的冷冻切片方法相结合，所描述的石蜡包埋方案有助于增强对NMJ的组织学分析。
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