Biobanking pp 299-311 | Cite as

An Introduction to Performing Immunofluorescence Staining

  • Kyuseok Im
  • Sergey Mareninov
  • M. Fernando Palma Diaz
  • William H. YongEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1897)


Immunofluorescence (IF) is an important immunochemical technique that allows for detection and localization of a wide variety of antigens in different types of tissues of various cell preparations. IF allows for excellent sensitivity and amplification of signal in comparison to immunohistochemistry, employing various microscopy techniques. There are two methods available, depending on the scope of the experiment or the specific antibodies in use: direct (primary) or indirect (secondary). Here, we describe preparation of specimens preserved in different types of media and step-by-step methods for both direct and indirect immunofluorescence staining.

Key words

Immunofluorescence Immunohistochemistry Immunocytochemistry Fixation Antigen retrieval Fluorescence Fluorophore 



This work was supported in part by NIH:NCI P50-CA211015, NIH:NIMH U24 MH100929, the Art of the Brain Foundation, and the Henry E. Singleton Brain Cancer Research Program.


  1. 1.
    Abcam (2008) Fixation and permeabilisation tips for IHC and ICC. Accessed 23 Feb 2016
  2. 2.
    Harlow E, Lane D (1999) Using antibodies: a laboratory manual. Cold Spring Harbor, NYGoogle Scholar
  3. 3.
    R&D Systems Inc. Antigen retrieval methods. Accessed 23 Feb 2016
  4. 4.
    Myers R. Technical brief of heat induced epitope retrieval. Accessed 24 Feb 2016
  5. 5.
    Myers J (2011) Overview of Heat-Induced Epitope Retrieval (HIER) techniques and devices. Accessed 24 Feb 2016
  6. 6.
    Vancells JC. Direct vs indirect immunofluorescence. Accessed 24 Feb 2016
  7. 7.
    Rockland Immunochemicals Inc. Immunofluorescence microscopy. Accessed 25 Feb 2016
  8. 8.
    Carpenter B. Secondary antibody selection guide. Accessed 25 Feb 2016
  9. 9.
    Abcam. A comparison between polyclonal and monoclonal. Accessed 26 Feb 2016
  10. 10.
  11. 11.
    Brelje TC, Wessendorf MW, Sorenson RL (1993) Multicolor laser scanning confocal immunofluorescence microscopy: practical application and limitations. Methods Cell Biol 38:97–181CrossRefGoogle Scholar
  12. 12.
    Thermo Scientific Inc (2012). Thermo scientific pierce fluorescent products guide. Accessed 29 Feb 2016
  13. 13.
    Cancer Institute, The University of Mississippi Medical Centre (2015) Selecting fluorochromes for analysis and sorting. Accessed 29 Feb 2016
  14. 14.
    Phillips ML (2007) How to maximize immunofluorescence multiplexing. Accessed 29 Feb 2016
  15. 15.
    Yarilin D, Xu K, Turkekul M et al (2015) Machine-based method for multiplex in situ molecular characterization of tissues by immunofluorescence detection. Nat Sci Rep.
  16. 16.
  17. 17.
    Shipitsin M, Small C, Giladi E et al (2014) Automated quantitative multiplex immunofluorescence in situ imaging identifies phospho-S6 and phospho-PRAS40 as predictive protein biomarkers for prostate cancer lethality. Proteome Sci 12:40. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Kyuseok Im
    • 1
    • 2
  • Sergey Mareninov
    • 1
    • 2
  • M. Fernando Palma Diaz
    • 1
  • William H. Yong
    • 1
    • 2
    • 3
    Email author
  1. 1.Department of Pathology and Laboratory MedicineDavid Geffen School of Medicine at UCLALos AngelesUSA
  2. 2.Brain Tumor Translational ResourceDavid Geffen School of Medicine at UCLALos AngelesUSA
  3. 3.Jonsson Comprehensive Cancer CenterDavid Geffen School of Medicine at UCLALos AngelesUSA

Personalised recommendations