Advancing Multiplexing in Immunofluorescence with Specific Secondary Antibodies from Jackson ImmunoResearch

blog / Immunology, Molecular Biology, News March 16 2023
Jackson immuno melanoma

Cells employ millions of proteins to perform cellular function. Thanks to the advance microscope techniques, we can visualize them by employing antibodies that specifically detects the protein of interest. Spatial proteomics have increased our understanding of protein localization in time and space thereby shedding lights on to its function in health, ageing and diseases.

We have previously discussed about Immunofluorescence (IF) technique and in the current blog we will focus on how Nordic Biosite together with Jackson Immuno Research helps you visualize proteins of interest simultaneously.IF detection of proteins can be either direct or indirect. In direct IF, a primary antibody tagged with a fluorescent molecule is employed to detect the protein of interest. Whereas, indirect IF depends on the use of a fluorescently conjugated secondary antibody that binds the primary antibody-protein complex. Indirect IF offers several advantages over direct IF including higher sensitivity, higher flexibility and is also cheaper. The downside of indirect IF is that secondary antibodies might cross react and have higher background noise as compared to direct IF. In addition, it is more time consuming and complex.  The limitation of sequential staining is overcome by multiplexing.  Multiplex Immunofluorescence (mIF) employs multiple primary antibodies targeting different epitopes or proteins that is later stained with secondary antibodies. mIF offers several advantages over traditional method as being highly efficient, reproducible, cost and time effective.

To perform successful mIF there are few things that one should take into consideration.

  • Make sure cells or tissue sections being analyzed are properly blocked using blocking reagents, BSA or normal serum. This ensures all unspecific binding is reduced thereby having reduced background signal.
  • Primary antibodies employed for detecting multiple proteins should be raised in different host.
  • Secondary antibodies targeting the primary antibody ideally should be from same host to reduce non-specific binding and background signal.
  • The conjugations on antibodies should be well selected. For example, fluorophores should not cross talk with each other as this will create signal bleed-through. This is done by ensuring the excitation and emission wavelength of the fluorophores are well separated from each other.


Biggest advantage of employing secondary antibody is the flexibility offered to researchers.  Jackson ImmunoResearch offers a wide spectrum of secondary antibodies that enables flexibility for IF like never before. Their product portfolio includes secondary antibodies raised in different host ranging from alpaca, bovine, donkey, goat, mouse, rabbit, rat and sheep. One can read more about the product portfolio here.

Jackson Immuno Research offers both IgG fraction and affinity purified donkey antibodies that can be used not only for mIF but for several applications including ELISA, WB and FC . The choice of affinity purified, or an IgG fraction depends on the research question and protein being studied. In cases where the protein under investigation is less abundant it is recommended to use IgG fraction since during the purification process high affinity antibodies can get bound to the matrix. On the other hand, affinity purified antibodies offers reduced background noise, low cross-reactivity and increased reproducibility since lot-to-lot variation is reduced. They also offer several choices to secondary antibody in terms of the available conjugates hence increasing your research horizon.


Nordic Biosite together with Jackson ImmunoResearch is proud to present you wide spectrum of secondary antibodies enabling efficient research. Find all the secondary antibodies from our partner

Discover Jackson Immunoresearch 

If you would like to learn more about the topic and how Nordic Biosite can help you with your research, please contact us at


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