Immunofluorescent analysis of (10% Formaldehyde) fixed HepG2 cells using 10427-2-AP (Calnexin antibody) at dilution of 1:50 and CoraLite®488-Conjugated Goat Anti-Rabbit IgG(H+L) (SA00013-2).
1X10^6 HepG2 cells were intracellularly stained with 0.2 ug Anti-Human Beta Actin (20536-1-AP) and CoraLite®488-Conjugated Goat Anti-Rabbit IgG(H+L) (SA00013-2) at dilution 1:1000 (green), and 0.2 ug Control Antibody. Cells were fixed with 90% MeOH.
Immunofluorescent analysis of HepG2 cells using 19842-1-AP (hIST1 antibody) at dilution of 1:25 and SA00013-4 CoraLite594-conjugated Goat Anti-Rabbit IgG(H+L).
1X10^6 HepG2 cells were intracellularly stained with 0.2 ug Anti-Human MYH9 (11128-1-AP) and Fluorescein (FITC)–conjugated Goat Anti-Rabbit IgG(H+L) (SA00003-2) at dilution 1:200 (red), and 0.2 ug Control Antibody. Cells were fixed with 90% MeOH.
Immunofluorescent analysis of (-20 ℃ Ethanol) fixed HepG2 cells using 66031-1-Ig (alpha Tubulin antibody) at dilution of 1:100 and CoraLite®488-Conjugated Goat Anti-Mouse IgG(H+L) (SA00013-1).
1X10^6 human peripheral blood lymphocytes were surface stained with 0.5 ug Anti-Human CD45 (65064-1-Ig, Clone: F10-89-4 ) and CoraLite®488-Conjugated Goat Anti-Mouse IgG(H+L) (SA00013-1) at dilution 1:1000 (green), or 0.5 ug Mouse IgG2a Isotype Control (black). Cells were not fixed.
1X10^6 HepG2 cells were stained with 0.2 ug Anti-Human MYH9 (11128-1-AP) and Cy3–conjugated Goat Anti-Rabbit IgG(H+L) (SA00009-2) at dilution 1:100 (red) Control Antibody in black. Cells were fixed with 4% PFA with 0.1 trition.
1X10^6 HeLa cells were stained with 0.2 ug Lamin B1 antibody (66095-1-Ig, red) and Fluorescein (FITC)–conjugated Goat Anti-Mouse IgG(H+L) (SA00003-1) with dilution 1:100. Control antibody (blue). Cell were fixed with 90% MeOH.
1X10^6 human peripheral blood lymphocytes were surface stained with 0.5 ug Anti-Human CD45 (65082-1-Ig, Clone: 2D1) and Cy3–conjugated Goat Anti-Mouse IgG(H+L) (SA00009-1) at dilution 1:50 (green), and 0.5 ug Control Antibody. Cells were not fixed.
Immunofluorescent analysis of fixed Hela cells using 10066-2-AP (HINFP antibody) at dilution of 1:25 and SA00007-2 (Rhodamine (TRITC)–conjugated Goat Anti-Rabbit IgG(H+L) (red). Blue pseudocolor = DAPI (fluorescent DNA dye).
Immunofluorescent analysis of MCF-7 cells, using B23 antibody 60096-1-lg at 1:25 dilution and SA00007-1 (Rhodamine (TRITC)–conjugated Goat Anti-Mouse IgG(H+L) (red). Blue pseudocolor = DAPI (fluorescent DNA dye).
Immunofluorescent analysis of (4% PFA) fixed paraffin-embedded mouse colon tissue using CD324 (E-cadherin) antibody (65241-1-Ig, Clone: DECMA-1 ) at dilution of 1:100 and Fluorescein (FITC)-conjugated Goat Anti-Rat IgG(H+L) SA00003-11. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0).
Immunofluorescent analysis of (4% PFA) fixed paraffin-embedded mouse colon tissue using CD324 (E-cadherin) antibody (65241-1-Ig, Clone: DECMA-1 ) at dilution of 1:100 and Fluorescein (FITC)-conjugated Goat Anti-Rat IgG(H+L) SA00003-11. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0).
1X10^6 NIH/3T3 cells were intracellularly stained with 0.5 ug Anti-Mouse CD107b (65052-1-Ig, Clone:ABL-93) and Fluorescein (FITC)-conjugated Goat Anti-Rat IgG(H+L) (SA00003-11) at dilution 1:50 (green), and 0.5 ug Control Antibody. Cells were fixed with 90% MeOH.
Well-defined and characterized immunostaining: Primary anti-rabbit IgG antibody (grey) with 2 copies each of a rabbit Fab- and Fc-specific monoclonal Nanobodies (green) bound. In total, 8 fluorophores (red stars) label the primary rabbit IgG antibody.
Higher resolution with anti-rabbit IgG Nano-Secondaries compared to conventional secondary antibodies: Left: Formation of a small, precise complex of Nanobodies (green) & primary antibody (grey). Right: Formation of a large, arbitrary complex of multiple polyclonal secondaries (green) & primary rabbit antibody.
Alpaca anti-rabbit IgG VHH Alexa Fluor® 488 was applied in conjunction with rabbit anti-Lamin B1 antibodies for immunostaining of nuclear lamina (green) in HeLa cells. Scale bar, 20 μm. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.
HeLa cells were immunostained with rabbit anti-Lamin B1 antibodies and alpaca anti-rabbit IgG VHH Alexa Fluor® 568 (1:1,000). Confocal and gated STED images were acquired with a Leica TCS SP8 STED 3X microscope, pulsed depletion with a 775 nm laser. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.
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 EGFP (EGFP-250, ChromoTek) added to HEK293T cell lysate. Detection with rabbit anti-GFP antibody PABG1 (ChromoTek) and alpaca anti-rabbit IgG VHH Alexa Fluor® 488.
Immunofluorescent analysis of (4% PFA) fixed HeLa cells using Alpha Tubulin antibody (66031-1-Ig, Clone: 1E4C11 ) at dilution of 1:400 and CoraLite®488-Conjugated AffiniPure Donkey Anti-Mouse IgG(H+L) (SA00013-5).
Immunofluorescent analysis of A431 cells, using Calnexin antibody 10427-2-AP at 1:50 dilution and SA00003-8 Fluorescein (FITC)–conjugated Donkey Anti-Rabbit IgG(H+L).
Human IgG (0.2 ug) were subjected to SDS PAGE followed by western blot with SA00003-12 at dilution of 1:1000 incubated at room temperature for 1.5 hours.
