Anti-mouse IgG2b Nano-Secondary: Well-defined and characterized immunostaining. Primary anti-mouse IgG2b antibody (grey) with 2X2 monoclonal mouse Fc- specific Nanobodies (green) bound. In total, 8 fluorophores (red stars) label the mouse IgG2b primary antibody.

Dissociation constant Kd and affinity.

Multiplexed immunostaining of HeLa cells with two alpaca anti-mouse Nano-Secondaries and one anti-rabbit Nano-Secondary. Green: mouse IgG1 anti-COX4 + alpaca anti-mouse IgG1 VHH Alexa Fluor® 488. Magenta: mouse IgG2b anti-Tubulin + alpaca anti-mouse IgG2b VHH Alexa Fluor® 647. Yellow: rabbit anti-Lamin + alpaca anti-rabbit IgG VHH Alexa Fluor® 568. Scale bar, 10 μm. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.

The anti-mouse IgG2b Nano-Secondary is subclass-specific and does not cross-react with IgGs from other commonly used species (here rabbit) and with mouse IgG1 and IgG3 subclasses.

One-step staining (left) vs. sequential staining (right) of HeLa cells with anti-Lamin A/C (nuclear lamina) mouse IgG2b monoclonal primary antibody + alpaca anti-mouse IgG2b VHH Alexa Fluor® 647 (magenta). Scale bar, 20 μm.

Multiplexed immunostaining of HeLa cells with two alpaca anti-mouse Nano-Secondaries and one anti-rabbit Nano-Secondary. Green: mouse IgG1 anti-COX4 + alpaca anti-mouse IgG1 VHH Alexa Fluor® 488. Yellow: rabbit anti-Lamin + alpaca anti-rabbit IgG VHH Alexa Fluor® 568. Magenta: mouse IgG2b anti-Tubulin + alpaca anti-mouse IgG2b VHH Alexa Fluor® 647. Scale bar, 10 μm. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.

One-step immunostaining is the simultaneous incubation of mouse IgG2b primary antibody and anti-mouse IgG2b Nano-Secondary. This method reduces incubation and hands-on time. Simultaneous incubation also supports multiplexing, tissue penetration, and cell staining for flow cytometry.

Multiple Nano-Secondaries can be applied for multiplex fluorescent Western blotting. This allows multiple targets to be analyzed simultaneously on the same blot at the same time. Multiplex fluorescent Western blot of GFP-TOM70, ®-Tubulin, and GFP in HEK293T cell lysate. Western blot membrane was simultaneously incubated with primary antibodies and Nano-Secondaries. Green: rabbit anti-GFP (ChromoTek PABG1) + alpaca anti-rabbit IgG VHH Alexa Fluor® 488. Magenta: mouse anti-®-Tubulin + alpaca anti-mouse IgG2b VHH Alexa Fluor® 647.

Western blot analysis of endogenous ®-Tubulin in HEK293T cell lysate. Detection with mouse anti-®-Tubulin antibody and alpaca anti-mouse IgG2b VHH Alexa Fluor® 647.

HeLa cells were immunostained with mouse IgG2b anti-®-Tubulin antibody + alpaca anti-mouse IgG2b VHH Alexa Fluor® 647 (magenta). Nuclei were stained with DAPI, blue. Scale bar, 10 μm. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.

Anti-mouse IgG2b Nano-Secondary: Well-defined and characterized immunostaining. Primary anti-mouse IgG2b antibody (grey) with 2X2 monoclonal mouse Fc- specific Nanobodies (green) bound. In total, 8 fluorophores (red stars) label the mouse IgG2b primary antibody.

One-step immunostaining is the simultaneous incubation of mouse IgG2b primary antibody and anti-mouse IgG2b Nano-Secondary. This method reduces incubation and hands-on time. Simultaneous incubation also supports multiplexing, tissue penetration, and cell staining for flow cytometry.

Dissociation constant Kd and affinity.

Anti-mouse IgG2b Nano-Secondary: Well-defined and characterized immunostaining. Primary anti-mouse IgG2b antibody (grey) with 2X2 monoclonal mouse Fc- specific Nanobodies (green) bound. In total, 8 fluorophores (red stars) label the mouse IgG2b primary antibody.

HeLa cells were immunostained with mouse IgG2b anti-βTubulin antibody + alpaca anti-mouse IgG2b VHH Alexa Fluor® 568 (red). Nuclei were stained with DAPI (blue). Scale bar, 10 μm. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich

One-step immunostaining is the simultaneous incubation of mouse IgG2b primary antibody and anti-mouse IgG2b Nano-Secondary. This method reduces incubation and hands-on time. Simultaneous incubation also supports multiplexing, tissue penetration, and cell staining for flow cytometry.

Dissociation constant Kd and affinity.

Immunofluorescence analysis of HeLa cells stained with mouse IgG2b anti-tubulin beta antibody and Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, CoraLite® Plus 488 (smsG2bCL488-1, green). Nuclei were stained with DAPI (blue). Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.

Immunofluorescence analysis of HeLa cells co-stained with mouse IgG2b anti-tubulin beta antibody and mouse IgG1 CD147 antibody followed by Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, CoraLite® Plus 488 (smsG2bCL488-1, green) and Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, CoraLite® Plus 647 (smsG1CL647-1, magenta). Nuclei were stained with DAPI (blue). Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.

HEK-293 cell lysates were subjected to SDS-PAGE followed by multiplex western blot analysis with 3 mouse primary antibodies including anti-Lamin B1 (66095-1-Ig), anti-HNRNPA (67445-1-Ig), and anti-Tom20 (66777-1-Ig). Primary antibodies were detected using 3 mouse IgG subclass-specific nano-secondary reagents including Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, CoraLite® Plus 647 (smsG1CL647-1, blue), Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 750 (smsG2aCL750-1, white), and Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, CoraLite® Plus 488 (smsG2bCL488-1, green).

Immunofluorescence analysis of HeLa cells stained with mouse IgG2b anti-Tubulin beta antibody and Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, CoraLite® Plus 555 (smsG2bCL555-1, orange). Nuclei were stained with DAPI (blue). Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.

HEK-293 cell lysates were subjected to SDS-PAGE followed by multiplex western blot analysis with 3 mouse primary antibodies including anti-ATP50 (66696-1-Ig), anti-AIF (67791-1-Ig), and anti-actin (66009-1-Ig). Primary antibodies were detected using 3 mouse IgG subclass-specific nano-secondary reagents including Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, CoraLite® Plus 750 (smsG1CL750-1, white), Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 488 (smsG2aCL488-1, green), and Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, CoraLite® Plus 555 (smsG2bCL555-1, red).

Immunofluorescence analysis of HeLa cells stained with mouse IgG2b anti-Lamin A/C antibody and Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, CoraLite® Plus 647 (smsG2bCL647-1, magenta). Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.

Immunofluorescence analysis of HeLa cells co-stained with mouse IgG1 anti-HSP60 antibody (66041-1-Ig) and mouse IgG2b anti-Lamin A/C antibody followed by Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, CoraLite® Plus 555 (smsG1CL555-1, yellow) and Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, CoraLite® Plus 647 (smsG2bCL647-1, magenta). Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.

HEK-293 cell lysates were subjected to SDS-PAGE followed by multiplex western blot analysis with 3 mouse primary antibodies including anti-PCNA (60097-1-Ig), anti-cytochrome C (66264-1-Ig), and anti-alpha tubulin (66031-1-Ig). Primary antibodies were detected using 3 mouse IgG subclass-specific nano-secondary reagents including Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, CoraLite® Plus 488 (smsG1CL488-1, green), Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 555 (smsG2aCL555-1, red), and Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, CoraLite® Plus 647 (smsG2bCL647-1, blue).