Overview of Immunofluorescence

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Immunofluorescence (IHC-IF) is an immunostaining technique that combines the use of antibodies with fluorescence imaging to visualize specific proteins and other biomolecules within a tissue sample. IHC-IF is a powerful technique that can be used with a variety of samples including fixed whole cells, paraffin-embedded cell pellets and frozen or paraffin-embedded tissue samples.

 

What is Immunofluorescence?

First described in 19411 and demonstrated in 19422 IHC-IF is an immunoassay technique that uses antibodies labeled either directly or indirectly with a fluorophore to visualize proteins and other biomolecules. The terms IHC-IF and immunohistochemistry (IHC) are often used interchangeably. However, both have grown to have distinct meanings, with IHC denoting the use of an enzymatic reaction resulting in a visible precipitate to visualize an antibody. Since its initial development, IHC-IF it has grown into a powerful tool enabling the visualization and colocalization of many proteins and biomolecules within a single tissue with an accompanying array of fluorophores that cover the entire visual spectrum.

 

How Does it Work?

IHC-IF protocols can be divided into three major steps: sample processing, staining, and imaging. Sample processing occurs prior to staining and typically involves fixing tissue samples in a cross-linking agent, such as paraformaldehyde, to preserve tissue morphology. Tissues are then dehydrated and embedded into paraffin blocks enabling tissues to be cut into tissue sections and affixed to a glass slide for long-term storage. When slides are ready to be stained they are processed in a clearing agent to remove excess paraffin and are rehydrated.

Staining protocols usually start with a blocking step consisting of either a protein or serum block to reduce non-specific binding. Sections are next incubated with an antibody specific to the protein or biomolecule of interest. At this point, there are two main types of IHC-IF protocols: direct and indirect. If the primary antibody is conjugated with a fluorophore then it is considered direct IHC-IF. An example of indirect IHC-IF is when the primary antibody is unconjugated and a targeted secondary antibody is conjugated with a fluorophore. Indirect IHC-IF has the advantage of increasing signal enabling the detection of lower abundant proteins, but at the cost of increased complexity and potential background.

Once slides are finished staining they are then imaged with either an epi-fluorescent, confocal, or multi-spectral microscope. Depending on the number and type of fluorophores used it may be necessary to do spectral unmixing to ensure that each marker is accurately quantitated. Although there is a plethora of fluorophores to choose from many have overlapping emission spectra limiting their use in multiplexing. Spectral unmixing is a powerful technique that distinguishes fluorophores not only from other fluorophores being used that have overlapping spectra but even background fluorescence.

 

Why Immunofluorescence

IHC-IF is a powerful technique when there is a need to visualize multiple proteins within a single sample. With advances in confocal microscopy and multispectral imaging it is now possible to stain more than 9 antigens in a single tissue, which is impossible with IHC. IHC-IF also allows users to understand co-expression and spatial relationship between multiple proteins and biomolecules through co-localization while still preserving tissue context that also is not possible with IHC.

 

Recent Immunofluorescence Citations Using Bethyl Antibodies

Bethyl sells a wide variety of high-quality antibodies for use in IHC-IF applications. These antibodies have recently been used to study:

  • The identification of telomerase as an aneuploidy survival factor in mammalian cells3
  • The identification of novel nesprin-1 binding partners4
  • The role of cathepsin B in lysosomal biogenesis and autophagy5
  • The licensing of primordial germ cells to gametogenesis-competent cells6
  • Transcription factors governing somatic and cancer stem cells7

 

Detection of human PD-L1 (red) and Lamin-A/C (green) in FFPE lung carcinoma by IHC-IF

Detection of human PD-L1 (red) and Lamin-A/C (green) in FFPE lung carcinoma by IHC-IF. Antibody: Rabbit anti-PDL1 recombinant monoclonal [BLR020E] (A700-020) and rabbit anti-Lamin-A/C (A303-430A). Secondary: DyLight® 594-conjugated goat anti-rabbit IgG (A120-201D4) and DyLight® 488-conjugated goat anti-rabbit IgG (A120-201D2). Counterstain: DAPI (blue).

Detection of human Beta-catenin (red) in FFPE lung carcinoma by IHC-IF

Detection of human Beta-catenin (red) in FFPE lung carcinoma by IHC-IF. Antibody: Rabbit anti-Beta-catenin (A302-010A). Secondary: DyLight® 594-conjugated goat anti-rabbit IgG (A120-201D4). Counterstain: DAPI (blue).

Detection of human CD20 (yellow) in FFPE tonsil by IHC-IF (pseudo color)

Detection of human CD20 (yellow) in FFPE tonsil by IHC-IF (pseudo color). Antibody: Mouse monoclonal anti-CD20 [L26] (A500-017A). Secondary: DyLight® 488-conjugated goat anti-rabbit IgG (A120-201D2). Counterstain: DAPI (blue).

 

Below is the entire list of targets involved in Immunofluorescence research. Can’t find what you are looking for? Bethyl offers a custom antibody service.

 

IF

T7
References

1. Coons A.H., Creech H.J., Jones R.N. 1941. Immunological properties of an antibody containing a fluorescent group. Experimental Biology and Medicine. 47(2):200-202.

2. Coons A.H., Creech, H.J., Jones R.N., Berliner, E. 1942. The demonstration of pneumococcal antigen in tissues by the use of fluorescent antibody. The Journal of Immunology. 45(3):159-170.

3. Meena J.K., Cerutti, A., Biechler, C., Morita, Y., Bruhn, C., Kumar M., Kraus J.M., Spiecher, M.R., Wang Z-Q., Kestler H.A., Fagagna F., Gunes C., Rudolph K.L. 2015. Telomerase abrogates aneuploidy-induced telomere replication stress, senescence and cell depletion. The EMBO Journal. 34: 1371-1384 [Bethyl antibody used: Phospho RPA32 (S33) (A300-246). Please note that this antibody has not been validated by Bethyl for use in IF and as such Bethyl cannot guarantee results as published in this paper.]

4. Rajgor D., Hanley J.G., Shanahan C.M. 2016. Identification of novel nesprin-1 binding partners and cytoplasmic matrin-3 in processing bodies. Molecular Biology of the Cell. 27(24):3894-3902. [Bethyl antibody used: DDX6 (A300-461A)]

5. Qi X., Ming Man S., Malireddi R.K., Karki R., Lupfer C., Gurung P., Neale G., Guy C.S., Lamkanfi M., Kanneganti T-D. 2016. Cathepsin B modulates lysosomal biogenesis and host defense against Francisella novicida infection. Journal of Experimental Medicine. 213(10):2081-2097. [Bethyl antibody used: TFEB (A303-673A). Please note that this antibody has not been validated by Bethyl for use in IF and as such Bethyl cannot guarantee results as published in this paper.]

6. H Y-C., Nicholls P.K., Soh Y.Q., Daniele J.R., Junker J.P., Oudenaarden A., Page D.C. 2015. Licensing of primordial germ cells for gametogenesis depends on genital ridge signaling. PLOS Genetics. 11(3):e1005019. [Bethyl antibody used: Nanog (IHC-00205). Please note that this antibody has not been validated by Bethyl for use in IF and as such Bethyl cannot guarantee results as published in this paper.]

7. Maddox J., Shakya A., South S., Shelton D., Anderson J.N., Chidester S., Kang J., Gligorich K.M., Jones D.A., Spangrude G.J., Welm B.E., Tantin D. 2012. Transcription factor Oct1 is a somatic and cancer stem cell determinant. PLOS Genetics. 8(11):e1003048. [Bethyl antibody used: OCT1 (A301-716A). Please note that this antibody has not been validated by Bethyl for use in IF and as such Bethyl cannot guarantee results as published in this paper.]