Molecular and Behavioral Neuroscience Institute, University of Michigan, 205 Zina Pitcher Place, Ann Arbor, MI, 48109, USA.
Department of Biological Sciences, Columbia University, New York, NY, USA.
Brain Struct Funct. 2018 Mar;223(2):987-999. doi: 10.1007/s00429-017-1583-z. Epub 2017 Dec 14.
The CLARITY technique enables three-dimensional visualization of fluorescent-labeled biomolecules in clarified intact brain samples, affording a unique view of molecular neuroanatomy and neurocircuitry. It is therefore, essential to find the ideal combination for clearing tissue and detecting the fluorescent-labeled signal. This method requires the formation of a formaldehyde-acrylamide fixative-generated hydrogel mesh through which cellular lipid is removed with sodium dodecyl sulfate. Several laboratories have used differential acrylamide and detergent concentrations to achieve better tissue clearing and antibody penetration, but the potential effects upon fluorescent signal retention is largely unknown. In an effort to optimize CLARITY processing procedures we performed quantitative parvalbumin immunofluorescence and lectin-based vasculature staining using either 4 or 8% sodium dodecyl sulfate detergent in combination with different acrylamide formulas in mouse brain slices. Using both confocal and CLARITY-optimized lightsheet microscope-acquired images, we demonstrate that 2% acrylamide monomer combined with 0.0125% bis-acrylamide and cleared with 4% sodium dodecyl sulfate generally provides the most optimal signal visualization amongst various hydrogel monomer concentrations, lipid removal times, and detergent concentrations.
CLARITY 技术能够对荧光标记的生物分子在经过澄清的完整脑组织样本中进行三维可视化，从而提供对分子神经解剖和神经回路的独特观察。因此，找到适合组织透明化和检测荧光标记信号的理想组合至关重要。该方法需要形成一种通过甲醛-丙烯酰胺固定剂生成的水凝胶网格，通过该网格用十二烷基硫酸钠去除细胞脂质。有几个实验室使用不同的丙烯酰胺和去污剂浓度来实现更好的组织透明化和抗体渗透，但荧光信号保留的潜在影响在很大程度上仍不清楚。为了优化 CLARITY 处理程序，我们使用 4%或 8%十二烷基硫酸钠去污剂与不同的丙烯酰胺配方组合，在小鼠脑切片上进行了定量钙结合蛋白免疫荧光和基于凝集素的血管染色。我们使用共聚焦和经过 CLARITY 优化的光片显微镜采集的图像，证明 2%丙烯酰胺单体与 0.0125%双丙烯酰胺结合，并使用 4%十二烷基硫酸钠进行透明化，通常在各种水凝胶单体浓度、脂质去除时间和去污剂浓度中提供了最佳的信号可视化效果。
