differential interference contrast, QD655 fluorescence, FLIPPER-body. is definitely fused to a nanobody against a target of interest. The modular nature of these probes allows an easy exchange of parts to change its target or color. A general FLIPPER-body focusing on GFP shows histone2B-GFP both in fluorescence and in EM. Similarly, endogenous EGF receptors and HER2 are visualized at nm-scale resolution in ultrastructural context. The small and flexible FLIPPER-body outperforms IgG-based immuno-labeling, likely by better reaching the epitopes. Given the modular domains and possibilities of nanobody generation for additional focuses on, FLIPPER-bodies have high potential to become a universal tool to identify proteins in immuno-CLEM with increased sensitivity compared to current methods. Electronic supplementary material The online version of this article (10.1007/s00418-018-1632-6) contains supplementary material, which is available to authorized users. varieties, consisting of a single chain, as opposed to IgGs (Hamers-Casterman et al. 1993; Helma et al. 2015). Genetic fusion of nanobodies with fluorescent proteins [FPs; chromobodies (Rothbauer et al. 2006)], as well as with ascorbate peroxidase 2 (APEX2) for EM visualization (Ariotti et al. 2015) have been used in co-expression systems and successfully display identification of several targets. However, co-expression in cells also affects PK68 protein function for some focuses on [(Helma et al. 2015); observe below]. Indirect EM labeling using an anti-nanobody antibody and a protein-A conjugated to platinum particles was recently PK68 pioneered, but still results in a relatively large range, ?17?nm, from your platinum particle to the prospective protein (HER2) (Kijanka et al. 2017). We reasoned that a solitary multi-domain protein that (1) can specifically target proteins based on a nanobody; (2) directly visualizes these using fluorescent proteins; and (3) identifies focuses on in EM using peroxidase-mediated DAB precipitation may have better labeling effectiveness than particle-based detection in EM. Since the architecture of the genetically-encoded probes allows easy website swapping, virtually unlimited mixtures between focusing on modules, FPs and peroxidase can be made (Fig.?1b). Note that, as opposed to most current genetically-encoded probes, our FLIPPER-bodies are 1st secreted by maker cells and consequently used, either like a supernatant or purified, like hybridoma generated monoclonal PK68 antibodies. Our results display that this protein-based probe highly efficiently labels focuses on, including in hard to reach subcellular areas, and outperforms current available alternatives. Methods Plasmids The sequences of the used FLIPPER-bodies are offered (Supplementary data). The backbones of the FLIPPER-bodies were ordered by Eurofins Genomics (Ebersberg, Germany) and modules were switched with standard molecular cloning tools. The transmission peptide originated from EpCAM. The cDNA of mCherry and dTomato [gifts from Roger Tsien; (Shaner et al. 2004)] were inserted using differential interference contrast, GFP fluorescence, QD655 fluorescence, FLIPPER-body, GFP and mCherry, ultrathin EM section. Bars LM and EM 10?m, EM zoom in 1?m. Unbiased large-scale high-resolution EM images are available via http://www.nanotomy.org Specific labeling of the EGFR by FLIPPER-bodies EGFR was chosen as the second target to optimize and verify FLIPPER-body technology. The new probe, consisting of a well-characterized anti-EGFR nanobody (7D12; Roovers et al. 2011), was positioned in the N-terminal of the FP and APEX2 modules (Fig.?1b). We switched the order, because binding capacity of the nanobody was lost after C-terminal conjugation. dTomato was chosen as FP because of its dimer-forming capacity that may double the fluorescence and EM signals. EGFR-GFP overexpressing cells were targeted with traditional immuno-CLEM using IgGs and QDs as explained above, as well as with anti-EGFR FLIPPER-body by pre-embedding labeling using non-permeabilized cells. The EGFR-GFP signal is present in the Golgi system as well as within the plasma membrane of the cells (Fig.?3). Traditional labeling clearly exposed QD fluorescence in the apical plasma membrane, but significantly less at cellCcell contact sites (Fig.?3a). Secondary antibodies conjugated with additional labels showed CD3D related results (Fig. S3). FLIPPER-body-mediated labeling of EGFR was clearly visible from the dTomato transmission as well as from the electron-dense staining (Fig.?3b). Note that the transmission was distributed equally over the surface of the cells, including at sites of cellCcell contacts that were not visualized with the traditional methods. Open in a separate windowpane Fig. 3 FLIPPER-body focuses on specific overexpressed EGFR. HEK293T expressing EGFR-GFP. a Detection of EGFR by GFP fluorescence, as well as by indirect labeling of EGFR with secondary antibodies conjugated to QD655. Note that QD655 are recognized in the periphery of the cells, but not at cellCcell contact sites. b Detection of EGFR using FLIPPER-body (cartoon right). The FLIPPER-body brands the EGFR-GFP positive cells exclusively, including on the cellCcell get in touch with sites. differential.