Anti-mouse IgG1 Nano-Secondary: Well-defined and characterized immunostaining. Primary anti-mouse IgG1 antibody (grey) with 2X2 monoclonal mouse Fc- specific Nanobodies (green) bound. In total, 8 fluorophores (red stars) label the mouse IgG1 primary antibody.

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

Vimentin in HeLa cells: The cells were stained with monoclonal anti-Vimentin mouse IgG1 antibody and alpaca anti-mouse IgG1 VHH Alexa Fluor® 488 and recorded with Leica GSDIM system. The image is a courtesy of Dr. Leila Nahidiazar, Dr. Jop Kind, and Prof. Kees Jalink.

One-step immunostaining is the simultaneous incubation of mouse IgG1 primary antibody and anti-mouse IgG1 Nano-Secondary. This method reduces incubation and hands-on time. Simultaneous incubation also supports multiplexing, tissue penetration, and cell staining for flow cytometry.

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.

Anti-mouse IgG1 Nano-Secondary: Well-defined and characterized immunostaining. Primary anti-mouse IgG1 antibody (grey) with 2X2 monoclonal mouse Fc- specific Nanobodies (green) bound. In total, 8 fluorophores (red stars) label the mouse IgG1 primary antibody.

One-step immunostaining is the simultaneous incubation of mouse IgG1 primary antibody and anti-mouse IgG1 Nano-Secondary. This method reduces incubation and hands-on time. Simultaneous incubation also supports multiplexing, tissue penetration, and cell staining for flow cytometry.

HeLa cells were immunostained with mouse IgG1 anti-αTubulin antibody + alpaca anti-mouse IgG1 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.

Anti-mouse IgG1 Nano-Secondary: Well-defined and characterized immunostaining. Primary anti-mouse IgG1 antibody (grey) with 2X2 monoclonal mouse Fc- specific Nanobodies (green) bound. In total, 8 fluorophores (red stars) label the mouse IgG1 primary antibody.

Immunostaining with Alpaca anti-mouse IgG1 Alexa Fluor 647 Nano-Secondaries for FACS. Jurkat CD3+ and Jurkat CD3- cell lines were mixed and immunostained live with anti-CD3 mouse IgG1 + alpaca anti-mouse IgG1 VHH Alexa Fluor 647 (1:600). CD3- cells were pre-stained with Calcein. Two cell populations can be clearly distinguished on the dot-plot: Alexa Fluor 647-positive Calcein-negative cells and Alexa Fluor 647-negative Calcein-positive cells.

Multiplexed immunostaining of HeLa cells with 3 subclass-specific alpaca anti-mouse Nano-Secondaries. Grey: Mouse IgG1 anti-Vimentin + alpaca anti-mouse IgG1 VHH Alexa Fluor® 647. Green: Mouse IgG2b anti-Lamin + alpaca anti-mouse IgG2b VHH Alexa Fluor® 488. Red: Mouse IgG3 anti-MOT + alpaca anti-mouse IgG3 VHH Alexa Fluor® 568. Scale bar, 10 μm. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.

Western blot analysis of EGFP (EGFP-250, ChromoTek) added to HEK293T cell lysate. Detection with anti-GFP mouse IgG 1 antibody and alpaca anti-mouse IgG1 VHH Alexa Fluor® 647.

One-step staining (left) vs. sequential staining (right) of HeLa cells with anti-COX4 (mitochondria) mouse IgG1 monoclonal primary antibody + alpaca anti-mouse IgG1 VHH Alexa Fluor® 647 (magenta). Cell nuclei are stained with DAPI (blue). Scale bar, 20 μm.

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

Multiplexed immunostaining of HeLa cells with 3 subclass-specific alpaca anti-mouse Nano-Secondaries. Grey: Mouse IgG1 anti-Vimentin + alpaca anti-mouse IgG1 VHH Alexa Fluor® 647. Green: Mouse IgG2b anti-Lamin + alpaca anti-mouse IgG2b VHH Alexa Fluor® 488. Red: Mouse IgG3 anti-MOT + alpaca anti-mouse IgG3 VHH Alexa Fluor® 568. Scale bar, 10 μm. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.

HeLa cells were immunostained with anti-Vimentin mouse IgG1 antibody and alpaca anti-mouse IgG1 VHH Alexa Fluor® 647 (grey). 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 IgG1 primary antibody and anti-mouse IgG1 Nano-Secondary. This method reduces incubation and hands-on time. Simultaneous incubation also supports multiplexing, tissue penetration, and cell staining for flow cytometry.

Flow cytometry with anti-mouse Nano-Secondaries. Jurkat CD3+ and Jurkat CD3- cell lines were mixed and immunostained live with anti-CD3 mouse IgG1 and alpaca anti-mouse IgG1 VHH Alexa Fluor® 647 (1:600). Two cell populations can be clearly distinguished with a 2-log shift: CD3-positive cells (in green) and CD3-negative cells (in grey).

Immunofluorescence analysis of HeLa cells stained with mouse IgG1 anti-vimentin antibody and Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, CoraLite® Plus 488 (smsG1CL488-1, green). 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-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).

Immunofluorescence analysis of HeLa cells stained with mouse IgG1 anti-HSP60 antibody (66041-1-Ig) and Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, CoraLite® Plus 555 (smsG1CL555-1, orange). 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 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 (smsG1CL647-1, 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-TDP43 (60019-1-Ig), anti-tubulin (66240-1-Ig), and anti-IMP3 (66247-1-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 555 (smsG1CL555-1, red), Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 647 (smsG2aCL647-1, blue), and Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, CoraLite® Plus 750 (smsG2bCL750-1, white).

Immunofluorescence analysis of HeLa cells stained with mouse IgG1 anti-CD147 antibody 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.

Immunofluorescence analysis of HeLa cells co-stained with mouse IgG2b anti-tubulin beta antibody and mouse IgG1 anti-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).