Imaging the Gut with "CLARITY".
Imaging the Gut with "CLARITY".
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DOI：
10.3791/62143
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发表时间：
2021-06-11
期刊：
影响因子：
--
通讯作者：
Neish AS
中科院分区：
文献类型：
--
作者：
Chandrasekharan B;Neish AS
CLARITY（透明脂质交换丙烯酰胺杂交刚性成像兼容组织水凝胶）最近发展成为一种有价值的技术，涉及丙烯酰胺包埋来降解组织（无需切片）并保留免疫染色的3-D组织结构。该技术与动态肠道环境的成像高度相关，在动态肠道环境中，不同类型的细胞在体内平衡和疾病状态下相互作用。本文描述了这种针对小鼠肠道进行优化的方法，它有助于追踪细胞类型，如上皮细胞、肠内分泌细胞、神经元、胶质细胞，以及神经元投射到上皮细胞或肠内分泌细胞中，分别介导微生物感知或营养化学感知。第1天，将肠道组织（1 - 1.5 cm）固定在4%多聚甲醛（PFA）中的磷酸盐缓冲盐水（PBS）中，在4℃下过夜。第2天，丢弃PFA，用PBS洗涤组织3次。在4°C下，将组织置于4%水凝胶（丙烯酰胺）溶液中的PBS（由30% protol稀释）中孵育过夜，以保持其完整性。第3天，将组织水凝胶溶液在37℃下孵育1小时，使水凝胶聚合。然后用PBS轻轻洗涤三次，去除多余的水凝胶。脱水（清除）的后续步骤包括组织在37°C的十二烷基硫酸钠（PBS中8% SDS）中在室温（RT）的摇床上孵育2天（第4天和第5天）。第6天，用PBS彻底清洗清理后的组织，去除SDS。组织可以用一抗（在含有0.3% Triton X-100的PBS中用0.5%正常驴血清稀释）孵育，在4°C下过夜，随后用适当的Alexa二抗在RT下孵育1.5小时，并用DAPI（1:10 000）进行核染色。将组织转移到干净的玻璃载玻片上，并使用vectasshield进行共聚焦成像。
CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) has recently evolved as a valuable technique involving acrylamide embedding to delipidate tissue (without sectioning) and to preserve the 3-D tissue structure for immunostaining. The technique is highly relevant in imaging the dynamic gut environment where different cell types interact during homeostasis and disease states. This method optimized for the mouse gut is described here, which helps to trace cell types like epithelia, enteroendocrine, neurons, glia, and the neuronal projections into the epithelia or enteroendocrine cells that mediate microbial sensing or nutrient chemo sensing respectively. The gut tissue (1–1.5 cm) is fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at 4 °C overnight on day 1. On day 2, PFA is discarded, and the tissue is washed thrice with PBS. The tissue is hydrogel embedded to preserve its integrity by incubation in 4% hydrogel (acrylamide) solution in PBS (diluted from 30% ProtoGel) overnight at 4 °C. On day 3, the tissue-hydrogel solution is incubated at 37 °C for 1 h to allow hydrogel polymerization. Tissue is then washed thrice gently with PBS to remove excess hydrogel. The subsequent step of delipidation (clearing) involves tissue incubation in sodium dodecyl sulfate (8% SDS in PBS) at 37 °C for 2 days (days 4 & 5) on a shaker at room temperature (RT). On day 6, the cleared tissue is thoroughly washed with PBS to remove SDS. Tissue can be immunostained by incubation in primary antibodies (diluted in 0.5% normal donkey serum in PBS containing 0.3% Triton X-100), overnight at 4°C, and subsequent incubation in appropriate secondary Alexa Fluor antibodies for 1.5 h at RT, and nuclear staining with DAPI (1: 10000). The tissue is transferred onto a clean glass slide and mounted using VectaShield for confocal imaging.
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