Immunofluorescence of MCF-7 cells: MCF-7 cells were fixed with 4% PFA and stained with Rabbit anti-ZO1 polyclonal antibody (21773-1-AP, 1:2000, green) and mouse anti-Alpha Tubulin monoclonal antibody (66031-1-Ig, 1:1000, magenta). Multi-rAb CoraLite® Plus 488-Goat Anti-Rabbit Recombinant Secondary Antibody (H+L) (RGAR002, 1:500) and Multi-rAb CoraLite® Plus 647-Goat Anti-Mouse Recombinant Secondary Antibody (H+L) (RGAM005, 1:500) were used for detection.

Immunofluorescence of Hela cells: Hela cells were fixed with 4% PFA and stained with Rabbit anti-Alpha Tubulin polyclonal antibody (11224-1-AP, 1:200, green) and mouse anti-NPM1 monoclonal antibody (60096-1-Ig, 1:1000, magenta). Multi-rAb CoraLite® Plus 488-Goat Anti-Rabbit Recombinant Secondary Antibody (H+L) (RGAR002, 1:500) and Multi-rAb CoraLite® Plus 647-Goat Anti-Mouse Recombinant Secondary Antibody (H+L) (RGAM005, 1:500) were used for detection.

Immunofluorescence of mouse cerebellum: mouse cerebellum FFPE section was stained with Rabbit anti-NeuN polyclonal antibody (26975-1-AP, 1:200, green) and mouse anti-Calbindin-D28k monoclonal antibody (66394-1-Ig, 1:200, magenta). Multi-rAb CoraLite® Plus 488 conjugated Recombinant Goat anti-rabbit secondary antibody (RGAR002, 1:500) and Multi-rAb CoraLite® Plus 647 conjugated Goat Anti-Mouse Recombinant Secondary Antibody (H+L) were used for detection (RGAM005, 1:500) .

Immunofluorescence of rat brain: rat brain FFPE section was stained with Rabbit anti-GFAP polyclonal antibody (16825-1-AP, 1:200, green) and mouse anti-NeuN monoclonal antibody (66836-1-Ig, magenta). Multi-rAb CoraLite® Plus 488 conjugated Recombinant Goat anti-rabbit secondary antibody (RGAR002, 1:500) and Multi-rAb CoraLite® Plus 647 conjugated Goat Anti-Mouse Recombinant Secondary Antibody (H+L) were used for detection (RGAM005, 1:500) .

1X10^6 MOLT4 were surface stained with 0.2 ug Anti-Human CD8 (65204-1-Ig, Clone: UCHT4) and Mouse IgG2a Isotype Control 66360-2-Ig. Multi-rAb CoraLite® Plus 647-Goat Anti-Mouse Recombinant Secondary Antibody (H+L) RGAM005 was used at 1:500 for detection.

Immunofluorescence of MCF-7 cells: MCF-7 cells were fixed with 4% PFA and stained with Rabbit anti-ZO1 polyclonal antibody (21773-1-AP, 1:2000, magenta) and mouse anti-Alpha Tubulin monoclonal antibody (66031-1-Ig, 1:1000, green). Multi-rAb CoraLite® Plus 647-Goat Anti-Rabbit Recombinant Secondary Antibody (H+L) (RGAR005, 1:500) and Multi-rAb CoraLite® Plus 488-Goat Anti-Mouse Recombinant Secondary Antibody (H+L) (RGAM002, 1:500) were used for detection.

Immunofluorescence of Hela cells: Hela cells were fixed with 4% PFA and stained with Rabbit anti-Alpha Tubulin polyclonal antibody (11224-1-AP, 1:200, magenta) and mouse anti-NPM1 monoclonal antibody (60096-1-Ig, 1:1000, green). Multi-rAb CoraLite® Plus 647-Goat Anti-Rabbit Recombinant Secondary Antibody (H+L) (RGAR005, 1:500) and Multi-rAb CoraLite® Plus 488-Goat Anti-Mouse Recombinant Secondary Antibody (H+L) (RGAM002, 1:500) were used for detection.

Immunofluorescence of mouse cerebellum: mouse cerebellum FFPE section was stained with Rabbit anti-NeuN polyclonal antibody (26975-1-AP, 1:200, magenta) and mouse anti-Calbindin-D28k monoclonal antibody (66394-1-Ig, 1:200, green). Multi-rAb CoraLite® Plus 647 conjugated Recombinant Goat anti-rabbit secondary antibody (RGAR005, 1:500) and Multi-rAb CoraLite® Plus 488 conjugated Goat Anti-Mouse Recombinant Secondary Antibody (H+L) were used for detection (RGAM002, 1:500) .

Immunofluorescence of rat brain: rat brain FFPE section was stained with Rabbit anti-GFAP polyclonal antibody (16825-1-AP, 1:200, magenta) and mouse anti-NeuN monoclonal antibody (66836-1-Ig, green). Multi-rAb CoraLite® Plus 647 conjugated Recombinant Goat anti-rabbit secondary antibody (RGAR005, 1:500) and Multi-rAb CoraLite® Plus 488 conjugated Goat Anti-Mouse Recombinant Secondary Antibody (H+L) were used for detection (RGAM002, 1:500) .

Immunofluorescence of Hela cells: Hela cells were fixed with 4% PFA and stained with Rabbit anti-GM130 polyclonal antibody (11308-1-AP, 1:200) Multi-rAb CoraLite® Plus 647 conjugated Goat Anti-Rabbit Recombinant Secondary Antibody (H+L) (RGAR005,1:800) was used for detection. Nucleus was stained with DAPI (blue).    The experiment was performed in Chromotek’s lab and image was recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.

1X10^6 MOLT4 were surface stained with 0.2 ug Anti-HLA class I rabbit polyclonal antibody (15240-1-AP) and Rabbit IgG Isotype Control 30000-0-AP. Multi-rAb CoraLite® Plus 647-Goat Anti-Rabbit Recombinant Secondary Antibody (H+L) RGAR005 was used at 1:500 for detection.

Immunofluorescent analysis of (4% PFA) fixed HeLa cells using 10594-1-AP (PEX14 antiboody) and CoraLite®647 conjugated AffiniPure F(ab')2 Fragment Goat Anti-Rabbit IgG (H+L) antibody (SA00014-9) at dilution of 1:150.

1X10^6 Jurkat cells were intracellularly stained with 0.2 ug Anti-human BCL2 Polyclonal antibody (12789-1-AP) and CoraLite®647-Conjugated AffiniPure F(ab')2 Fragment Goat Anti-Rabbit IgG (H+L) (SA00014-9) at dilution 1:1000 (red), and 0.2 ug Control Antibody. Cells were fixed with 4% PFA and permeabilized with Flow Cytometry Perm Buffer (PF00011-C).

Immunofluorescent analysis of (-20°C Ethanol) fixed HeLa cells using 12987-1-AP (Lamin B antibody) and the CoraLite®647-Mouse Anti-Heavy chain of Rabbit IgG antibody (SA00014-6), at dilution of 1:200.

1X10^6 HepG2 cells were intracellularly stained with Anti-human TDP-43 recombinant antibody (80002-1-RR) and 0.2 ug CoraLite®647 Mouse Anti-Rabbit Heavy chain of Rabbit IgG (SA00014-6) (red), or 0.2 ug Control Antibody. Cells were fixed and permeabilized with Transcription Factor Staining Buffer Kit (PF00011).

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.

Immunofluorescent analysis of (4% PFA) fixed HeLa cells using 10594-1-AP (PEX14 antiboody) and CoraLite®647 conjugated AffiniPure F(ab')2 Fragment Donkey Anti-Rabbit IgG (H+L) antibody (SA00014-7) at dilution of 1:150.

1X10^6 HEK-293 cells were intracellularly stained with 0.2 ug Anti-GOLGA2/GM130 Polyclonal antibody (11308-1-AP) and CoraLite®647-Conjugated AffiniPure F(ab')2 Fragment Donkey Anti-Rabbit IgG (H+L) (SA00014-7) at dilution 1:1000 (red), or 0.2 ug Control Antibody. Cells were fixed with 4% PFA and permeabilized with Flow Cytometry Perm Buffer (PF00011-C).

Immunofluorescent analysis of (4% PFA) fixed HeLa cells using 66009-1-Ig (Beta Actin antiboody) and CoraLite®647 conjugated AffiniPure F(ab')2 Fragment Goat Anti-Mouse IgG (H+L) antibody (SA00014-10) at dilution of 1:150.

1X10^6 HepG2 cells were intracellularly stained with 0.2 ug Anti-human RBAP48 Monoclonal antibody (66060-1-Ig) and CoraLite®647-Conjugated AffiniPure F(ab')2 Fragment Goat Anti-Mouse IgG (H+L) (SA00014-10) at dilution 1:1000 (red), and 0.2 ug Control Antibody. Cells were fixed with 4% PFA and permeabilized with Flow Cytometry Perm Buffer (PF00011-C).

Immunofluorescent analysis of (4% PFA) fixed HeLa cells using 66009-1-Ig (Beta Actin antiboody) and CoraLite®647 conjugated AffiniPure F(ab')2 Fragment Donkey Anti-Mouse IgG (H+L) antibody (SA00014-8) at dilution of 1:150.

1X10^6 HepG2 cells were intracellularly stained with 0.2 ug Anti-human RBAP48 Monoclonal antibody (66060-1-Ig) and CoraLite®647-Conjugated AffiniPure F(ab')2 Fragment Donkey Anti-Mouse IgG (H+L) (SA00014-8) at dilution 1:1000 (red), and 0.2 ug Control Antibody. Cells were fixed with 4% PFA and permeabilized with Flow Cytometry Perm Buffer (PF00011-C).