HeLa cells transiently expressing Mannosidase II-EGFP were immunostained with GFP recombinant antibody, VHH-mouse IgG1 Fc fusion [CTK0201] (gfms, 1:2,000) and Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, Alexa Fluor® 647 [CTK0103, CTK0104] (sms1AF647-1, 1:500). Scale bar, 10 μM.

HeLa cells transiently expressing Mannosidase II-EGFP were immunostained with GFP recombinant antibody, VHH-rabbit IgG Fc fusion [CTK0201] (gfrb-20, 1:1,000) and Nano-Secondary? alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, Alexa Fluor? 647 [CTK0101, CTK0102] (srbAF647-1, 1:1,000). Scale bar, 10 μM.

Structure of VHH.

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).

Immunofluorescence analysis of HeLa cells stained with mouse IgG1 anti-B23 antibody and Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, CoraLite® Plus 750 (smsG1CL750-1, white). Nuclei were stained with DAPI (blue).

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).

Schematic representation of anti-mouse IgG2a Nano-Secondary binding to its target. Mouse IgG2a primary antibody (grey) is bound with two identical monoclonal mouse IgG2a Fc- specific Nanobody molecules (green). In total, 4 fluorophores (red stars) label the mouse IgG2a primary antibody.

Dot-blot assay demonstrates that anti-mouse IgG2a Nano-Secondary is subclass-specific and does not cross-react with other mouse IgG subclasses including IgG1, IgG2b, IgG2c and IgG3.

The alpaca anti-mouse IgG2a Fc-specific recombinant VHH [CTK0114] has a very high affinity (KD <10 pM) for mouse IgG2a primary antibody.

Immunofluorescence analysis of Hela cells stained with mouse IgG2a anti-Tubulin antibody (66240-1-Ig) and Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 647 (magenta). Nuclei were stained with DAPI (blue). Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.

Flow cytometry analysis of 1X10^6 human peripheral blood mononuclear cells (PBMCs) stained with anti-human CD3 (clone OKT3, 65133-1-Ig) and Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 647.