Several tissue clearing methods have been developed for three-dimensional imaging of thick specimens. Here, we applied CUBIC and ScaleS approaches to whole-mounted vaginal wall to reveal spatial distribution of γδ T lymphocytes, the key cells engaged in the epithelial homeostasis control and immune surveillance. Both methods rendered the tissue transparent and enabled detection of the green fluorescent protein (GFP)-expressing γδ T cells in vaginal samples of Tcrd-H2BeGFP transgenic mice. Upon additional immunolabeling, however, only CUBIC preserved the GFP signal and allowed for cell localization assessment during the estrous cycle. Using a combination of single- and two-photon microscopy, we found that during the diestrus phase the number of γδ T cells in the vaginal wall increased compared to estrus, while the proportion of cells residing in epithelium and stroma remained constant, irrespective of the cycle phase, and was close to 3:1, respectively. Moreover, the distance from epithelial γδ T cells to laminin-positive basal membrane and collagen-rich stroma also increased in diestrus in spite of thinning of epithelium upon shedding cornified cells. Our data indicate that γδ T cells sense sex hormone fluxes which influence their number and position them closer to the vaginal lumen in the diestrus phase.

中文翻译：

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更深入：使用组织清除方法对小鼠生殖道中γδT细胞进行三维研究。

已经开发了几种用于清除厚样本的三维图像的组织清除方法。在这里，我们将CUBIC和ScaleS方法应用于整个阴道壁，以揭示γδT淋巴细胞的空间分布，γδT淋巴细胞是参与上皮稳态控制和免疫监视的关键细胞。两种方法均使组织透明，并能够检测Tcrd-H2BeGFP转基因小鼠阴道样品中表达绿色荧光蛋白（GFP）的γδT细胞。然而，在进行额外的免疫标记后，只有CUBIC保留了GFP信号，并允许在发情周期内进行细胞定位评估。结合使用单光子和双光子显微镜，我们发现在发情期，与发情期相比，阴道壁中的γδT细胞数量增加了，而细胞的比例在上皮细胞和间质中保持不变，与周期阶段无关，分别接近3：1。此外，尽管脱落的角质化细胞使上皮变薄，但从上皮开始，从上皮γδT细胞到层粘连蛋白阳性基底膜和富含胶原的基质的距离也增加了。我们的数据表明，γδT细胞感觉到性激素通量，该通量会影响性激素通量，并使它们在雌激素阶段更靠近阴道腔。