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.

Immunofluorescent analysis of (4% PFA) fixed OCT-embedded frozen mouse small intestine tissue using smooth muscle actin antibody (14395-1-AP) at dilution of 1:400 and Multi-rAb CoraLite ® Plus 647-Goat Anti-Rabbit Recombinant Secondary Antibody (H+L) (RGAR005).

Immunofluorescent analysis of (4% PFA) fixed OCT-embedded frozen mouse colon tissue using E-cadherin antibody (20874-1-AP) at dilution of 1:400 and Multi-rAb CoraLite ® Plus 647-Goat Anti-Rabbit Recombinant Secondary Antibody (H+L) (RGAR005).

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.

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.

Live A431 cells were incubated with anti-human EGFR (Cetuximab biosimilar) followed by Nano-Secondary® alpaca anti-human IgG, recombinant VHH, CoraLite® Plus 647 [CTK0117] (magenta, shuGCL647-2 ). Cells were fixed and nuclei were stained with DAPI (blue). Epifluorescence images were acquired with a 40x objective and post-processed.

Immunofluorescence analysis of transgenic CHO-TIM3 cells. PFA-fixed cells were stained with anti-human TIM3 (Cobolimab biosimilar), Nano-Secondary® alpaca anti-human IgG, recombinant VHH, CoraLite® Plus 647 [CTK0117] (gray) and DAPI (blue).

Flow cytometry analysis of 1X10^6 human peripheral blood mononuclear cells (PBMCs) stained with anti-human CD20 ( Rituximab biosimilar) and Nano-Secondary® alpaca anti-human IgG, recombinant VHH, CoraLite® Plus 647 [CTK0117) (shuGCL647-2). Cells were co-stained with CoraLite® Plus 488 conjugated anti-human CD19 antibody.

Dot blot assay demonstrates that anti-human IgG Nano-Secondary (CTK0117) has no cross-reactivity to human IgA/IgM/IgE, and goat, guinea pig, mouse, rabbit, rat, and sheep serum. It shows slight cross-reactivity to macaque serum.

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

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.

Immunofluorescent analysis of (4% PFA) fixed OCT-embedded frozen mouse heart tissue using ACTC1-specific antibody (66125-1-Ig, Clone: 1F2B9 ) at dilution of 1:800 and Multi-rAb CoraLite ® Plus 647-Goat Anti-Mouse Recombinant Secondary Antibody (H+L) (RGAM005).

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 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).

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.

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).

HEK-293 and Hela cell lysates were subjected to SDS-PAGE followed by fluorescent western blot analysis with rabbit anti-GAPDH antiboy (10494-1-AP) and Nano-Secondary® alpaca anti-rabbit IgG, recombinant VHH, CoraLite® Plus 647 (srb2GCL647-1), 1:500.

Immunofluorescence analysis of A431 cells stained with rabbit anti-COXIV antibody (11242-1-AP, magenta) and Nano-Secondary® alpaca anti-rabbit IgG, recombinant VHH, CoraLite® Plus 647 (srb2GCL647-1), 1:500. Nuclei were stained with DAPI (blue). Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.

Dot blot assay demonstrates that both clones contributing to Nano-Secondary® alpaca anti-rabbit IgG, recombinant VHH CoraLite Plus 647 [CTK0119, CTK0120] have no cross-reactivity to mouse, rat, or human immunoglobulins.