For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome

We use cookies to make our site as useful as possible.

Our Cookie Policy explains how you can opt-out of the cookies we use.

If you continue without changing your cookie settings, we'll assume you’re happy with this.

Continue Continue

United States
Your country/region is currently set to:

If incorrect, please enter your country/region into the box below, to view site information related to your country/region.

Call (888) 77-ABCAM (22226) or contact us
Need help? Contact us

  • My account
  • Sign out
Sign in or Register with us

Welcome

Sign in or

Don't have an account?

Register with us
My basket
Quick order
Abcam homepage

  • Research Products
    By product type
    Primary antibodies
    Secondary antibodies
    ELISA and Matched Antibody Pair Kits
    Cell and tissue imaging tools
    Cellular and biochemical assays
    Proteins and Peptides
    By product type
    Proteomics tools
    Agonists, activators, antagonists and inhibitors
    Cell lines and Lysates
    Multiplex miRNA assays
    Multiplex Assays
    By research area
    Cancer
    Cardiovascular
    Cell Biology
    Epigenetics
    Metabolism
    Developmental Biology
    By research area
    Immunology
    Microbiology
    Neuroscience
    Signal Transduction
    Stem Cells
  • Diagnostic & Therapeutic Solutions
    Custom solutions & partnerships

    Custom antibody development and commercial partnerships to advance your diagnostic and therapeutic discovery.

    Create custom solutions with us

    Partner with us

  • Support
    Support hub

    Access advice and support for any research roadblock

    View support hub

    Protocols

    Your experiments laid out step by step

    View protocols

  • Events
    • Conference calendar
    • Cancer
    • Cardiovascular
    • Epigenetics & Nuclear signaling
    • Immunology
    • Neuroscience
    • Stem cells
    • Tradeshows
    • Scientific webinars
    Keep up to date with the latest events

    Full event breakdown with abstracts, speakers, registration and more

    View global event calendar

  • Pathways
    Cell signalling pathways

    View all pathways

    View all interactive pathways

Supporting our customers and employees during the COVID-19 pandemic. Read more

Double and triple immunostaining using secondary antibodies

Related

  • Alexa Fluor® secondary antibodies
    • Alexa Fluor® 488 conjugated secondaries
      • Alexa Fluor® 594 conjugated secondaries
        • Alexa Fluor® 647 conjugated secondaries
          • Pre-adsorbed secondary antibodies
              • Cell imaging
                • Direct vs indirect IF
                  • Fluorochrome chart
                    • Compare Alexa Fluor®488 to FITC
                      • Secondary antibody selection guide
                          • Western blot tools
                            • Infrared fluorescence vs chemiluminescence

                              These simple tips will minimize background staining in your experiments as well as prevent false positives or negatives.

                              Double or triple labeling using indirect methods commonly uses primary antibodies raised in different species. Although multiple labeling with primary antibodies raised in the same species is also possible. The primary antibodies are then visualized with secondary antibodies conjugated to different fluorophores. Here are some essential tips you need to remember for your double or triple stainings when using secondary antibodies:

                              1. Ideally, all secondary antibodies should come from the same host species. Use blocking serum from the same species in which the secondaries have been raised to avoid non-specific binding and reduce background.

                              2. Use pre-adsorbed secondary antibodies to minimize species cross-reactivity. If possible, select secondary antibodies that have been pre-adsorbed against the species of the experimental sample as well as the host species of the other primaries.

                              3. Use the brightest fluorophores for low abundant proteins and vice versa to maximize sensitivity. Fluorescent conjugates vary in their brightness and antigens vary in their expression levels. Highly expressed antigens will be resolved with almost any fluorophore, but antigens expressed at lower levels may require brighter fluorophores such as R-phycoerythrin.

                              4. Use fluorophores with narrow emission spectra to avoid bleed-through (also known as crossover or crosstalk). Narrow bandpass filters come at the cost of lower signal levels. But even with narrow bandpass filters, emission from one fluorophore can bleed through the detection channel intended for another.


                              Our Alexa Fluor® secondary antibodies range offers nine different dyes with minimal spectral overlap, while covering the whole spectrum. The table below shows the compatibility between the nine Alexa Fluor® dyes we offer.

                              The compatibility between the different Alexa Fluor® dyes is indicated as high (green), medium (blue) or low (red). High compatibility indicates no crosstalk between fluorophores. Pairs of fluorophores with low compatibility should be avoided in the same experiment as much as possible due to high cross-talk between them.

                              Alexa Fluor® dyes are the brightest and most photostable labels available. Our Alexa Fluor® conjugated secondaries are:

                              • Manufactured in our laboratories: complying with high quality standards that guarantee optimal performance.
                              • Validated in cellular imaging: each product batch is tested in immunofluorescence using confocal microscopy to ensure consistency.
                              Explore our range of Alexa Fluor® secondaries


                              As an example, below is an image of a double immunostaining experiment using secondary antibodies following the recommendations above.

                              Image: Courtesy of Dr. Shaohua Li, UMDNJ-Robert Wood Johnson Medical School

                              Sample: mouse embryonic stem cell-differentiated embryoid bodies (EBs)

                              Counterstain: DAPI

                              AntibodiesAntigen A - LamininAntigen B - Ki67
                              Primary AntibodyRabbit polyclonal to laminin α1Mouse monoclonal to Ki67
                              Secondary Antibody

                              Goat anti-rabbit IgG H&L (Alexa Fluor® 594) preadsorbed (ab150084)

                              Minimal cross-reactivity with Chicken, Cow, Horse, Human, Pig, Rabbit, and Rat.

                              Goat anti-mouse IgG H&L (Alexa Fluor® 488) preadsorbed (ab150117)

                              Minimal cross-reactivity with Human, Mouse, and Rat.

                              Note: In this example both secondaries have been raised in goat, and hence, they do not recognize each other. The secondaries have also been pre-adsorbed so that they do not recognize the other primary antibodies as well as endogenous mouse immunoglobulins that may be present in the tissue.


                              Alexa Fluor® is a registered trademark of Life Technologies.  Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.

                              Get resources and offers direct to your inbox Sign up
                              A-Z by research area
                              • Cancer
                              • Cardiovascular
                              • Cell biology
                              • Developmental biology
                              • Epigenetics & Nuclear signaling
                              • Immunology
                              • Metabolism
                              • Microbiology
                              • Neuroscience
                              • Signal transduction
                              • Stem cells
                              A-Z by product type
                              • Primary antibodies
                              • Secondary antibodies
                              • Biochemicals
                              • Isotype controls
                              • Flow cytometry multi-color selector
                              • Kits
                              • Loading controls
                              • Lysates
                              • Peptides
                              • Proteins
                              • Slides
                              • Tags and cell markers
                              • Tools & Reagents
                              Help & support
                              • Support
                              • Make an Inquiry
                              • Protocols & troubleshooting
                              • Placing an order
                              • RabMAb products
                              • Biochemical product FAQs
                              • Training
                              • Browse by Target
                              Company
                              • Corporate site
                              • Investor relations
                              • Company news
                              • Careers
                              • About us
                              • Blog
                              Events
                              • Tradeshows
                              • Conferences
                              International websites
                              • abcam.cn
                              • abcam.co.jp

                              Join with us

                              • LinkedIn
                              • facebook
                              • Twitter
                              • YouTube
                              • Terms of sale
                              • Website terms of use
                              • Cookie policy
                              • Privacy policy
                              • Legal
                              • Modern slavery statement
                              © 1998-2021 Abcam plc. All rights reserved.