一种高效、快速的小鼠肾小球标记和分析方法
Summary
本研究提出了一套易于使用、完整且简单的方法来标记和分析来自 CUBIC 清除的小鼠肾脏的肾小球。使用异硫氰酸荧光素 （FITC）-葡聚糖、光片荧光显微镜 （LSFM） 或常见的共聚焦显微镜和软件（如 Imaris）可以轻松可靠地获得肾小球数量和体积等数据。
Abstract
肾小球是肾脏的基本单位;因此，研究肾小球对于了解肾功能和病理学至关重要。生物成像提供直观的信息;因此，标记和观察肾小球具有重要意义。然而，目前使用的肾小球观察方法需要复杂的操作，并且结果可能会丢失标签细节或三维（3D）信息。清晰、畅通无阻的脑成像鸡尾酒和计算分析（CUBIC）组织清除技术已广泛应用于肾脏研究，可实现更准确的检测和更深的检测深度。我们发现，通过尾静脉注射中等分子量的FITC-葡聚糖，然后采用CUBIC透明化方法，可以快速有效地标记小鼠肾小球。清除的小鼠肾脏可以通过光片显微镜（或切片时的共聚焦显微镜）扫描，以获得整个肾脏中所有肾小球的三维图像堆栈。使用适当的软件处理，肾小球信号可以很容易地数字化并进一步分析，以测量肾小球的数量、体积和频率。
Introduction
肾小球的数量和体积对于各种肾脏疾病的诊断和治疗非常重要1,2,3,4,5。肾小球数估计的黄金标准是物理解剖器/分馏器组合。然而，这种方法需要特殊的试剂和设备，使其速度慢且昂贵6,7,8,9。活检提供了丰富的信息，但显然，这种方法只适用于粗略估计10,11。医学成像技术，包括磁共振成像（MRI）、计算机断层扫描（CT）和X射线，也广泛用于肾小球检测12,13,14,15，但这些技术需要笨重的仪器。新方法，如基质辅助激光解吸/电离 （MALDI） 成像质谱仪16 或厚薄切片法17，也已用于肾小球检测，尽管它们仍然繁琐而费力。
在透明技术的帮助下，可以观察更深的深度，并从厚组织甚至整个器官中获得更丰富、更完整的信息18,19,20,21,22,23。因此，透明技术在肾脏研究中得到了广泛的应用24。还涉及清除肾脏中肾小球的观察和检测。然而，这些已发表的文章要么只是简单地提到肾小球检测25，要么使用难以实现的标记方法，如转基因动物26、自产染料13或高浓度抗体孵育27来标记肾小球。此外，尽管研究分析了清除肾脏中的肾小球，但分析总是有限的13或依赖于作者自己建立的分析算法26。
我们之前已经展示了一种更方便的方法来标记小鼠肾脏中的肾小球28。通过使用 Imaris，我们发现可以快速获得肾小球计数、频率和体积。因此，在这里，我们提出了一套更易于访问、更全面和更简化的方法来标记和分析小鼠肾脏的肾小球。
Protocol
本研究使用成年C57BL / 6小鼠（6周龄，25-30g）。所有程序均按照当地动物福利和实验伦理法规进行。该研究已获得四川大学华西医院生物医学研究伦理委员会的批准。 1. 肾小球标记和组织制备 肾小球标记将FITC-葡聚糖（10mg）以1：1（1mg：1mL）的比例溶解在1x磷酸盐缓冲盐水（PBS）中，以制备工作探针溶液。注意：工作溶液可以在4°C下储存1个月。</l…
Representative Results
本研究为小鼠肾脏肾小球的标记和分析提供了一种简单有效的方法。 肾小球（血管）可以通过血管内注射 FITC-葡聚糖很好地标记。清除过程后，肾脏变得透明（图1A），并且可以通过使用光片显微镜（图1B）或共聚焦显微镜（图1C）清楚地观察到肾小球。共聚焦显微镜的扫描深度有限，因此应将肾脏切成约 1 ?…
Discussion
组织清除技术可分为3组或4组29,30,31。有机溶剂型组织透明化（例如DISCO和PEGASOS）、水性组织透明化（例如CUBIC）和水凝胶包埋组织透明化（例如CLARITY）均已应用于肾脏透明化25,26,28,32。正如我们已经证明的那样，当肾小球（血管）…
Divulgations
The authors have nothing to disclose.
Acknowledgements
本研究得到了国家自然科学基金（82204951）和四川省科学技术计划（2020JDRC0102）的资助。
Materials
| 4% PFA | Biosharp | 7007171800 | Fixation reaagen |
| 502 Glue | Deli | 7146 | For fixing the kidney to the sample fixing adapter |
| Antipyrine | Aladdin | A110660 | Clearing reagent |
| Brain Matrix | RWD Life Science | 1mm 40-75 | Tissue slicing |
| Confocal microscopy | Nikon | A1plus | Image acquisition |
| FITC-Dextran | Sigma-Aldrich | FD150S | Labeling reagent |
| Light sheet fluorescence microscopy | Zeiss | Light sheet 7 | Image acquisition |
| Mice | Ensiweier | Adult C57BL/6 mice (6 weeks of age, 25–30 g) | |
| N-Butyldiethanolamine | Aladdin | B299095 | Clearing reagent |
| Nicotinamide | Aladdin | N105042 | Clearing reagent |
| Pentobarbital Natriumsalz | Sigma-Aldrich | P3761 | |
| Tail vein fixator | JINUOTAI | JNT-FS35 | Fix the mouse for vail injection |
| Triton X-100 | Sigma-Aldrich | T8787 | Clearing reagent |
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Citer Cet Article
Bai, L., Wu, Y., Dai, W., Shi, Q., Wu, L., Zhang, J., Zheng, L. An Efficient and Fast Method for Labeling and Analyzing Mouse Glomeruli. J. Vis. Exp. (204), e65973, doi:10.3791/65973 (2024).