Purpose

We have previously used Immuno Tomography (IT) to identify label-retaining stem cell populations in the cornea and meibomian gland. While this method provides the unique ability to quantify stem cell populations comprised of 1–4 cells, the number of antigens that can be sequentially used to characterize these unique cells is limited by antigen stability after antibody stripping and re-probing. To address this deficiency, we have evaluated the capability of Imaging Mass Cytometry™ (IMC™) to generate multiplexed images using metal-conjugated antibodies to label IT plastic sections and generate 3-dimensional IMC data sets (3D IMC).

Methods

K5–H2B-GFP mice, 56 days after doxycycline chase, were sacrificed and eyelid tissue processed for IT. A total of 400 serial, plastic sections, 2 μm thick, were then probed using metal-tagged antibodies specific for sox 9, collagen type I, E-cadherin, Ki67, GFP, αSMA, vimentin, and DNA intercalator. Multiplexed images were then generated using an Imaging Mass Cytometry system (Fluidigm®), and 3D reconstructions were assembled.

Results

All 8 metal-labeled tags were detected and their images were successfully assembled into 3D IMC data sets. GFP-labeled nuclei were identified within the meibomian glands in comparable numbers to those previously reported for slow-cycling meibomian gland stem cells.

Conclusions

These findings demonstrate that IMC can be used on plastic sections to generate multiplexed, 3D data sets that can be reconstructed to show the spatial localization of meibomian gland stem cells. We propose that 3D IMC might prove valuable in more fully characterizing stem cell populations in different tissues.

中文翻译：

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免疫断层扫描 (IT) 和成像质谱流式细胞术 (IMC)，用于构建空间分辨的多重 3D IMC 数据集

目的

我们之前曾使用免疫断层扫描 (IT) 来识别角膜和睑板腺中保留标记的干细胞群。虽然该方法提供了量化由 1-4 个细胞组成的干细胞群的独特能力，但可连续用于表征这些独特细胞的抗原数量受到抗体剥离和重新探测后抗原稳定性的限制。为了解决这一缺陷，我们评估了 Imaging Mass Cytometry™ (IMC™) 使用金属缀合抗体生成多重图像来标记 IT 塑料切片并生成 3 维 IMC 数据集 (3D IMC) 的能力。

方法

强力霉素追踪后 56 天，处死 K5–H2B-GFP 小鼠，并处理眼睑组织用于 IT。然后使用 sox 9、I 型胶原蛋白、E-钙粘蛋白、Ki67、GFP、αSMA、波形蛋白和 DNA 嵌入剂特异性金属标记抗体对总共 400 个 2 μm 厚的连续塑料切片进行探测。然后使用成像质量细胞计数系统 (Fluidigm®) 生成多重图像，并组装 3D 重建。

结果

所有 8 个金属标记标签均被检测到，并且它们的图像已成功组装成 3D IMC 数据集。在睑板腺内鉴定出 GFP 标记的细胞核，其数量与之前报道的慢循环睑板腺干细胞的数量相当。

结论

这些发现表明，IMC 可用于塑料切片，生成多重 3D 数据集，这些数据集可重建以显示睑板腺干细胞的空间定位。我们认为 3D IMC 在更全面地表征不同组织中的干细胞群方面可能具有价值。