Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping.
Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping.
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DOI：
10.3390/ijms22168644
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发表时间：
2021-08-11
影响因子：
5.6
通讯作者：
Guo J
中科院分区：
文献类型：
--
作者：
Pham T;Nazaroff CD;Labaer J;Guo J
对天然细胞环境中的蛋白质进行多重单细胞分析，对于揭示复杂生物系统中不同细胞类型的组成、相互作用和功能具有巨大的希望。然而，现有的多重蛋白质成像技术受到其检测灵敏度或技术要求的限制。为了解决这些问题，在这里，我们开发了一种超灵敏和多重原位蛋白质分析方法，通过用现成的抗体和可切割的荧光酪胺（CFT）进行重复染色。在该方法的每个循环中，蛋白质靶标被用辣根过氧化物酶标记的抗体识别，辣根过氧化物酶催化CFT在蛋白质靶标上或附近的共价沉积。成像后，荧光团被化学切割，抗体被剥离。通过染色、成像、荧光团切割和抗体剥离的连续循环，可以在单个细胞中原位定量大量蛋白质。应用这种方法，我们分析了人类福尔马林固定石蜡包埋（FFPE）扁桃体组织中约67，000个细胞中每个细胞中的20种不同蛋白质。基于其独特的蛋白质表达谱和微环境，这些单个细胞被划分为不同的细胞簇。我们还通过检查哪些特定的细胞簇选择性地相互关联或避免彼此来探索组织中的细胞-细胞相互作用。
Multiplexed single-cell analysis of proteins in their native cellular contexts holds great promise to reveal the composition, interaction and function of the distinct cell types in complex biological systems. However, the existing multiplexed protein imaging technologies are limited by their detection sensitivity or technical demands. To address these issues, here, we develop an ultrasensitive and multiplexed in situ protein profiling approach by reiterative staining with off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this approach, the protein targets are recognized by antibodies labeled with horseradish peroxidase, which catalyze the covalent deposition of CFT on or close to the protein targets. After imaging, the fluorophores are chemically cleaved, and the antibodies are stripped. Through continuous cycles of staining, imaging, fluorophore cleavage and antibody stripping, a large number of proteins can be quantified in individual cells in situ. Applying this method, we analyzed 20 different proteins in each of ~67,000 cells in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Based on their unique protein expression profiles and microenvironment, these individual cells are partitioned into different cell clusters. We also explored the cell–cell interactions in the tissue by examining which specific cell clusters are selectively associating or avoiding each other.
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下丘脑前区域的分子，空间和功能性单细胞分析。
DOI：
10.1126/science.aau5324
发表时间：
2018-11-16
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Moffitt JR;Bambah-Mukku D;Eichhorn SW;Vaughn E;Shekhar K;Perez JD;Rubinstein ND;Hao J;Regev A;Dulac C;Zhuang X
通讯作者：
Zhuang X
影响因子：
9.8
作者：
McQuin C;Goodman A;Chernyshev V;Kamentsky L;Cimini BA;Karhohs KW;Doan M;Ding L;Rafelski SM;Thirstrup D;Wiegraebe W;Singh S;Becker T;Caicedo JC;Carpenter AE
通讯作者：
Carpenter AE
影响因子：
64.5
作者：
Goltsev Y;Samusik N;Kennedy-Darling J;Bhate S;Hale M;Vazquez G;Black S;Nolan GP
通讯作者：
Nolan GP
影响因子：
5.5
作者：
Liao R;Mondal M;Nazaroff CD;Mastroeni D;Coleman PD;Labaer J;Guo J
通讯作者：
Guo J
影响因子：
16.6
作者：
Piehowski, Paul D.;Zhu, Ying;Burnum-Johnson, Kristin E.
通讯作者：
Burnum-Johnson, Kristin E.