The process is repeated until DNA-PAINT images for each target protein are acquired. a characterization method for recombinant viral proteins on both cells and VLPs. We were able to quantify the amount of the three main influenza proteins (hemagglutinin (HA), neuraminidase (NA), and ion channel matrix protein 2 (M2)) per cell and per VLP with nanometer resolution IQ 3 and single-molecule sensitivity, proving that DNA-PAINT is a powerful technique to characterize recombinant viral constructs. times, and in our case, it was repeated 3 times to image the 3 target proteins sequentially. (5) After image acquisition, analysis and postprocessing of the image reveal a density map for each protein target. To do so, we preliminarily prepared secondary antibodies labeled with a DNA-docking strand, following the protocol from Schnitzbauer et al.45 (Table S1). Each secondary antibody paired approximately 2.5 docking strains, obtaining comparable amounts of localizations (between 8 and 10 localizations per single antibody, Figure S11). We then immunostained each protein of interest (HA, M2, and NA) with a specific primary antibody and the corresponding secondary antibody, bearing a docking strand. In this way, each protein is labeled with docking strands having a specific oligonucleotide sequence. Finally, the super-resolution DNA-PAINT images were acquired by adding the imager, a DNA strand conjugated with Atto-647N, so that each imager targets the secondary antibody exposing its complementary docking strand. Since the three imagers employed in this study bear the same dye (Atto-647N) and only differ in the oligonucleotide sequence, they were introduced sequentially to perform multiplexed imaging of the three Tgfb2 proteins. As illustrated in Figure ?Figure11B, the first DNA-PAINT image of a target protein is acquired upon introduction of the corresponding imager in solution; subsequently, the solution is exchanged and replaced with one containing another imager, so that a different target protein is imaged with DNA-PAINT. The process is repeated until DNA-PAINT pictures for each focus on protein are obtained. A chamber-tubing program linked to a pump was utilized to switch solutions as the test was preserved in the same placement (Amount S1). Finally, a fake color was designated to each obtained DNA-PAINT picture sequentially, as well as the pictures had been merged to reconstruct your final multicolor DNA-PAINT picture of the three focus on protein. To recognize the timeline from the post-transfection (p.t.) appearance of recombinant protein, we checked the expression after 24 and 48 h p initial.t. with fluorescence microscopy (Amount S2). Results verified that after 24 h the appearance levels were comparable to those at 48 h p.t., which may be the best time point where VLPs are harvested in the culture medium.14,18 Thus, to monitor the recombinant IQ 3 proteins expression quantitatively, DNA-PAINT pictures from the three protein over the cell membrane were obtained at both 24 and 48 h p.t., using the same imager focus (1.5 nM) to acquire comparable results. Amount ?Amount22A,B displays representative super-resolution pictures obtained for every proteins (M2, HA, NA) in the same area from the membrane. Aesthetically, the appearance degree of the three protein seemed very similar at both 24 h p.t. (Amount ?Amount22A) and 48 h p.t. (Amount ?Figure22B). To be able to evaluate appearance amounts for the various protein quantitatively, in each picture, the density from the DNA-PAINT localizations was calculated in a certain area corresponding towards the cell. Open in another window Amount 2 DNA-PAINT imaging on cells expressing recombinant protein after 24 or 48 h post transfection (p.t.). DNA-PAINT super-resolution pictures of cell expressing the three influenza proteins (HA, NA, and M2 as well as the mergence from the three of these) after (A) 24 h p.t. and (B) 48 p.t. The images show an certain section of the cell basal membrane. Scale club: 5 m. (C) Quantification from the localization thickness of HA, NA, and M2 at 24 h and 48 h p.t. per cell (= 10; one stage, one cell). Blue: 24 h p.t.; crimson: 48 h p.t. SD and Mean. (D) Viral proteins localizations from the three recombinant protein expressed inside the same cell IQ 3 (= 10; one stage, one cell). Blue: 24 h p.t.; crimson: 48 h p.t. The axes HA, NA,.