c2 laser scanning confocal microscope  (Nikon)

Journal: Viruses

Article Title: Neutralizing and Enhancing Epitopes of the SARS-CoV-2 Receptor-Binding Domain (RBD) Identified by Nanobodies

doi: 10.3390/v15061252

Figure Lengend Snippet: Purified 6× His-tagged nanobodies of E. coli clones 103, 105, 114, and 278 (VH103, VH105, VH114, and VH278, respectively) and their antigen binding: ( A ) Purified nanobodies from inclusion bodies of the vh -pET23b+ plasmid-transformed NiCo21 (DE3) E. coli clones 103, 105, 114, and 278 (lanes 1–4, respectively). M, protein standard marker. The numbers on the left are protein masses in kDa. ( B ) Indirect ELISA for testing the binding of 6× His-tagged VH103, VH105, VH114, and VH278 to recombinant Wuhan, Delta, and Omicron RBDs, using BSA as a control antigen. ( C – E ) The 6× His-tagged VH103, VH105, VH114, and VH278, respectively, bound to native S1 subunits of spike proteins of SARS-CoV-2 Wuhan wildtype and Delta and Omicron variants, as determined by confocal microscopy. Nanobodies stained red; native S1 subunits of the SARS-CoV-2 spike protein stained green; nuclei stained blue; co-localized VHs and S1 subunits in merged panels stained orange/yellow. ( F ) Half-maximal effective concentrations (EC50) of VH103, VH105, VH114, and VH278 against the recombinant S1 subunit.

Article Snippet: Secondary antibodies, including 1:400 dilutions of Alexa Fluor Plus 488 goat anti-rabbit IgG (Invitrogen) and Alexa Fluor Plus 555 goat anti-mouse IgG (Invitrogen), were added to the cells and kept at 4 °C for 1 h. The cells were washed with PBS, and their nuclei were stained with DAPI (Invitrogen) for 10 min. After washing, the cover slips were mounted, and the cells were examined using laser scanning confocal microscopy (Nikon C2+ Eclipse Ti2-E Laser Confocal Microscope, Nikon, Melville, NY, USA).

Techniques: Purification, Clone Assay, Binding Assay, Plasmid Preparation, Transformation Assay, Marker, Indirect ELISA, Recombinant, Control, Confocal Microscopy, Staining