The voltage-gated sodium channel [pore-forming subunit of the neuronal voltage-gated sodium channel (NaV1.6)] has recently been found in cardiac myocytes. Emerging studies indicate a role for NaV1.6 in ionic homeostasis as well as arrhythmogenesis. Little is known about the spatial organization of these channels in cardiac muscle, mainly due to the lack of high-fidelity antibodies. Therefore, we developed and rigorously validated a novel rabbit polyclonal NaV1.6 antibody and undertook super-resolution microscopy studies of NaV1.6 localization in cardiac muscle. We developed and validated a novel rabbit polyclonal antibody against a C-terminal epitope on the neuronal sodium channel 1.6 (NaV1.6). Raw sera showed high affinity in immuno-fluorescence studies, which was improved with affinity purification. The antibody was rigorously validated for specificity via multiple approaches. Lastly, we used this antibody in proximity ligation assay (PLA) and super-resolution STochastic Optical Reconstruction Microscopy (STORM) studies, which revealed enrichment of NaV1.6 in close proximity to ryanodine receptor (RyR2), a key calcium (Ca2+) cycling protein, in cardiac myocytes. In summary, our novel NaV1.6 antibody demonstrates high degrees of specificity and fidelity in multiple preparations. It enabled multimodal microscopic studies and revealed that over half of the NaV1.6 channels in cardiac myocytes are located within 100 nm of ryanodine receptor Ca2+ release channels.

中文翻译：

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使用新型高保真抗体的超分辨率成像显示神经元钠通道 NaV1.6 与心肌中的瑞安定受体密切相关

电压门控钠通道 [神经元电压门控钠通道 (Na五1.6)] 最近在心肌细胞中发现。新兴研究表明钠的作用五1.6 在离子稳态以及心律失常发生中。关于心肌中这些通道的空间组织知之甚少，主要是由于缺乏高保真抗体。因此，我们开发并严格验证了一种新型兔多克隆钠五1.6 抗体并进行了Na的超分辨率显微镜研究五1.6 在心肌中的定位。我们开发并验证了一种针对神经元钠通道 1.6（Na五1.6）。原始血清在免疫荧光研究中显示出高亲和力，亲和纯化得到改善。通过多种方法对抗体的特异性进行了严格验证。最后，我们将该抗体用于邻近连接测定 (PLA) 和超分辨率随机光学重建显微镜 (STORM) 研究，结果显示 Na 富集五1.6 紧邻兰尼碱受体（RyR2），一种关键的钙（Ca2+) 循环蛋白，在心肌细胞中。总之，我们的小说Na五1.6 抗体在多种制剂中表现出高度的特异性和保真度。它使多模态微观研究成为可能，并揭示了超过一半的 Na五心肌细胞中的 1.6 个通道位于兰尼碱受体 Ca 的 100 nm 范围内2+发布渠道。