Rfp booster atto594

Primary antibodies anti-EEA1 (ab70521), anti-58K Golgi (ab27043), antipericentrin (ab28144), and anticatalase (ab110292) were purchased from Abcam, anti-TGN46-8 (SAB4200355) from Sigma and anti-Tom20 (sc-17764) from Santa Cruz Biotechnology. All primary antibodies were monoclonal mouse antibodies. Polyclonal secondary antimouse antibodies conjugated to Atto647N (50185, Sigma Aldrich) as well as isotype-specific secondary antimouse antibodies conjugated to Atto647N (610-156-041, Rockland Immunochemicals) and Alexa Fluor 568 (A-21124, Thermo Fisher Scientific) were used for detection. The nanoboosters—single-domain alpaca antibody fragments covalently coupled to fluorescent dyes—GFP-Booster Atto488 and RFP-Booster Atto594 (Chromotek) were used to boost eYFP and mCherry fluorescence, respectively. Live cell stains SiR-tubulin, SiR-actin, and SiR-lysosome were from Spirochrome and MitoTracker Deep Red FM was from Thermo Fisher Scientific.

As primary antibodies we used the HPV16-L1 detecting antibody 16L1-312F (mouse monoclonal, diluted 1:200; [29 (link)]) and a rabbit polyclonal antibody raised against the V5-tag (diluted 1:5000; cat# ab9116, Abcam, Cambridge, UK). In addition, we employed GFP-Booster Atto488 (Chromotek, cat# gba488, Planegg-Martinsried, Germany) and RFP-Booster Atto594 (cat# rba594, Chromotek, Planegg-Martinsried, Germany), both diluted 1:200. As secondary antibodies we used AlexaFluor488 labelled donkey anti-mouse (cat# A-21202, Carlsbad, CA) and AlexaFluor594 labelled donkey anti-rabbit (cat# ab150064, Abcam). For transfection, we used the previously described plasmids encoding CD63-GFP [18 (link)], V5-OBSL1 [27 (link), 30 (link)] and CD151-RFP [31 (link)].