Cyclic Immunofluorescence (CycIF), A Highly Multiplexed Method for Single‐cell Imaging

• DOI: 10.1002/cpch.14
• Publication Date: 2016-12-15T00:51:47Z
• Authors: Jia‐Ren Lin; Mohammad Fallahi‐...; Jia‐Yun Chen; Peter K. Sorger
• Keywords: Immunofluorescence; Alexa Fluor; Multiplex

Cyclic Immunofluorescence (CycIF) is a public-domain method for performing highly multiplexed immunofluorescence imaging using conventional epifluorescence microscope. It uses simple reagents and existing antibodies to construct images with up 30 channels by sequential 4- 6-channel followed fluorophore inactivation. Three variant methods are described, the most generally useful of which involves staining fixed cells directly conjugated Alexa Fluor dyes in four colors, inactivating fluorophores mild base presence hydrogen peroxide light, then another round imaging. Cell morphology preserved through multiple rounds CycIF, signal-to-noise ratios appear increase. Unlike antibody-stripping methods, CycIF gentle optimized monolayers cultured cells. A second protocol indirect third enables chemical inactivation genetically encoded fluorescent proteins, allowing multiplex be combined live-cell analysis expressing reporter proteins. © 2016 John Wiley & Sons, Inc.