Double and triple immunostaining using secondary antibodies

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 (orange) 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.

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.

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