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"I have used a wide variety of secondaries and Jackson ImmunoResearch has consistently been the best. The fluorophores are bright and stable and their selective (x reactivity removed) secondaries have always shown species specificity in multiple labeling."
Janet Duerr, Ohio University
Immunohistochemistry (IHC) is a powerful technique, indispensable in research and in clinical diagnostics. It is a staple of the pathology lab for disease diagnostics and classification. In research, IHC is used to interrogate many biological processes, including visualization of protein expression patterns, characterizing protein interactions, and identification of tissue boundaries. The researcher can thereby observe the distribution and localization of specific structures within the context of the cellular architecture.
Triple immunofluorescence. Jejunum villus probed for Ulex - Biotin using Cy™5 Streptavidin (blue 016-170-084), GIP by Cy™2 AffiniPure Donkey Anti-Goat IgG (H+L) (green 705-225-147), tubulin by Cy™3 AffiniPure Donkey Anti-Mouse IgG (H+L) (red 715-165-150 ). Image courtesy of Brian McAdams and William Kennedy, University of Minnesota.
A tissue analyte is specifically recognized by a primary antibody, which may be directly conjugated (direct IHC); or the primary may itself be detected by a conjugated secondary antibody (indirect IHC), allowing visualization of the analyte. The conjugated antibody can be labeled with any of several reporter molecules, including enzymes, fluorophores and colloidal gold. The expression of multiple analytes can be observed and characterized by individual primary/secondary antibody pairs in multiple labeling protocols.
Analytes of interest may be detected on the surface of the tissue or interrogated internally through permeabilization of the sample. The analyte (antigen) can be detected directly (A), or indirectly (B) using a fluorescent (Immunofluorescence) or reporter enzyme (colorimetric) probe conjugated to a secondary antibody.
The Indirect method using a secondary antibody offers many advantages. It preserves the antigen binding site on the primary antibody by conjugating to the secondary antibody. Since secondary antibodies are available conjugated to a wide variety of fluorophores, the indirect method enhances the possibilities for multiple labeling. In addition, it provides inherent signal enhancement, whereby multiple secondary antibodies bind to one antigen-bound primary antibody. Signal can be further enhanced by using a biotinylated secondary antibody followed by conjugated streptavidin (C), or by using the peroxidase-anti-peroxidase (PAP) method.
A: Direct IHC, B: Indirect IHC, C: Signal enhancement with PAP
Recommended dilution ranges for Histo-/Cyto-chemistry are shown in the table below.
|Unconjugated whole IgG and F(ab') 2 secondary antibodies||10-20 µg / ml|
|All Alexa Fluor®, DyLight, and Cy3 Conjugates [IgG, F(ab') 2, Fab]||1:100 - 1:800|
|AMCA, Cy2, FITC, TRITC, RRX, and Texas Red Conjugates [IgG, F(ab') 2, Fab]||1:50 - 1:200|
|Cy5 Conjugates [IgG, F(ab') 2, Fab]||1:100 - 1:400|
|Horseradish Peroxidase Conjugates [IgG, F(ab') 2]||1:500 - 1:5,000|
|Biotin-SP Conjugates[IgG, F(ab') 2] (using Fluorophore-Conjugated Streptavidin)||1:200 - 1:1,000|
|Biotin-SP Conjugates [IgG, F(ab') 2] (using enzyme-Conjugated Streptavidin)||1:500 - 1:5,000|
|Horseradish Peroxidase-Conjugated Streptavidin||1-2 µg / ml|
|Alkaline Phosphatase-Conjugated Streptavidin||1-2 µg / ml|
|All Alexa Fluor®-, DyLight 405-, and Cy3-Conjugated Streptavidin||0.5-2 µg / ml|
|Cy5-Conjugated Streptavidin||0.5-2 µg / ml|
|All Other Fluorophore-Conjugated Streptavidin||2-5 µg / ml|
Primary cultures of neonatal rat cardiomyocytes stained with Cy3-AffiniPure Goat Anti-Mouse IgG (H+L) [Cat# 115-165-146] and Cy2-AffiniPure Goat Anti-Rabbit IgG (H+L) [Cat# 111-225-144] secondary antibodies together with DAPI counterstain. Image courtesy of Elisabeth Ehler, KCL’
“When labeling for more than one target with antibodies from closely related species, you really need to certain your secondary antibody is specific!”
Read on to find out how Cardiac researcher Elisabeth Ehler uses Jackson ImmunoResearch cross-adsorbed (min X) secondary antibodies to multiple label with primary antibodies derived from closely related host species – enabling her to differentiate cell types in primary cardiac cell cultures.
JIR provides Alexa Fluor 488, 594, 647, 680, 790 conjugates.
Alexa Fluor® fluorescent dyes are widely recognized as superior fluorescent dyes, respected for their brightness and photostability. They are suitable for a variety of applications including confocal microscopy, flow cytometry, fluorescent Western blotting, and fluorescent ELISA. Alexa Fluor conjugated secondary antibodies can be used exclusively or in combination with other fluorescent probes to generate an optimal multiplex dye panel for your experimental variables.
Possible uses of FabuLights also include labeling cell surface immunoglobulins without cross-linking and activating B cells, and labeling Fc chimeras (fusion proteins).
FabuLight antibodies are Fab fragment secondary antibodies specific to the Fc region of IgG or IgM primary antibodies.
They are available conjugated with 9 different fluorophores and biotin, and enable labeling of primary antibodies prior to incubation with cells or tissue.
They provide a time saving alternative to sequential incubation flow cytometry and immunohistochemistry procedures, without compromising the active site of the primary antibody.