Jackson Immuno Research Inc.
specializing in secondary antibodies and conjugates

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Whole IgG Affinity-Purified Secondary Antibodies

"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

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Secondary Antibodies

For VHH Discovery

Anti-Alpaca IgG, Subclass and VHH domain secondaries
  • Ideally time PBMC harvest
  • Enhance detection of VHH antibodies
  • Test for VHH expression and binding activity

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Secondary Antibodies for Immunohistochemistry - Immunofluorescence

Secondary Antibodies for Immunohistochemistry (IHC)

Introduction to IHC

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.

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Triple immunofluorescence image 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.

Principles of Immunohistochemistry

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.

Direct or Indirect Immunohistochemistry

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.

Direct and Indirect Detection Methods A: Direct IHC, B: Indirect IHC, C: Signal enhancement with PAP

Multiplex IHC

Chromogenic Detection for Western Blot, IHC, and ELISA

Cell and Tissue Staining with JIR Secondary Antibodies

Recommended dilution ranges for Histo-/Cyto-chemistry are shown in the table below.

Product Histo-/Cyto-Chemistry
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

Featured products

Neonatal Rat Cardiomyocytes Stained with Cross-adsorbed JIR Secondaries 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’

Distinguish cell types from primary cultures

“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.

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Jackson ImmunoResearch Alexa Fluor Secondary Antibodies JIR provides Alexa Fluor 488, 594, 647, 680, 790 conjugates.

Alexa Fluor 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.

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# Possible uses of FabuLights also include labeling cell surface immunoglobulins without cross-linking and activating B cells, and labeling Fc chimeras (fusion proteins).

Label Primary Antibodies In Solution Without Compromising Activity

FabuLight™ - Fab Anti-Fc Fragment Secondary Antibodies

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.

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