Anti-mouse IgG3 Nano-Secondary: Well-defined and characterized immunostaining. Primary anti-mouse IgG3 antibody (grey) with 2 copies of a monoclonal mouse Fab- specific Nanobody (green) bound. In total, 6 fluorophores (red stars) label the mouse IgG3 primary antibody.

HeLa cells were immunostained with mouse IgG3 anti-Lamin A/C antibody + alpaca anti-mouse IgG3 VHH Alexa Fluor® 647 (magenta). Scale bar, 10 μm. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.

One-step staining (left) vs. sequential staining (right) of HeLa cells with anti-MOT (mitochondria) mouse IgG3 monoclonal primary antibody + alpaca anti-mouse IgG3 VHH Alexa Fluor® 647 (magenta). Cell nuclei are stained with DAPI (blue). Scale bar, 20 μm.

Multiplexed immunostaining of HeLa cells with two alpaca anti-mouse Nano-Secondaries and one conventional secondary antibody. Green: mouse IgG3 anti-Lamin + alpaca anti-mouse IgG3 VHH Alexa Fluor® 488. Red: mouse IgG1 anti-Tubulin + alpaca anti-mouse gG1 VHH Alexa Fluor® 568. Magenta: rabbit anti-Ki67 + conventional polyclonal secondary anti-rabbit-AF647. Scale bar, 10 μm. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.

Higher resolution with anti-mouse IgG3 Nano-Secondary compared to conventional secondary antibodies. Left: Formation of a small, precise complex of Nanobodies (green) & primary antibody (grey). Right: Formation of a large, arbitrary complex of multiple polyclonal secondaries (green) & primary antibody.