Illuminating the Invisible: A Comprehensive Guide to Immunofluorescence

Michael Hernando^*
*Correspondence: Michael Hernando, Department of Medical Oncology, Vall D’Hebron University Hospital, Barcelona, Spain, Email:

Introduction

In the world of biomedical research and diagnostics, scientists have harnessed the power of light to uncover hidden mysteries within cells and tissues. One of the most valuable techniques in this regard is immunofluorescence, a method that allows researchers to visualize and study specific molecules within biological samples. This groundbreaking technique has revolutionized our understanding of cellular and molecular biology, enabling us to explore a wide range of scientific questions and clinical applications. In this comprehensive guide to immunofluorescence, we will delve into the principles, methods, applications and future prospects of this indispensable tool. Immunofluorescence is a powerful imaging technique that combines the specificity of antibodies with the illuminating properties of fluorescent dyes. It allows researchers to pinpoint and visualize the presence and distribution of specific proteins, nucleic acids, or other molecules within cells or tissues [1].

Description

By attaching fluorescent molecules to target molecules of interest, researchers can "light up" these molecules, making them visible under a fluorescence microscope. Before delving deeper into immunofluorescence, it's essential to understand the fundamental principles of fluorescence. When certain molecules, called fluorophores, are exposed to light of a specific wavelength, they absorb the light energy and re-emit it at a longer wavelength. This phenomenon is known as fluorescence. Fluorophores come in various colors, each emitting light of a unique color when excited by a specific wavelength of light. In immunofluorescence, fluorophores are attached to antibodies or other molecules that can bind specifically to the target of interest. When these fluorophore-labeled molecules bind to their targets within a sample, the target becomes fluorescent and can be detected and imaged using a fluorescence microscope [2].

Direct immunofluorescence and indirect immunofluorescence. Direct immunofluorescence involves the use of a single antibody labeled with a fluorescent dye. This antibody directly binds to the target molecule of interest in the sample. While direct immunofluorescence is relatively straightforward, it may not provide as much signal amplification as the indirect method. Indirect immunofluorescence is a more commonly used technique. It involves two primary antibodies: the primary antibody and the secondary antibody [3]. The primary antibody specifically binds to the target molecule, as in direct immunofluorescence. However, instead of directly attaching the fluorescent dye to the primary antibody, a secondary antibody is used. This secondary antibody is conjugated with a fluorescent dye and binds specifically to the primary antibody [4,5].

Conclusion

This indirect approach allows for signal amplification because multiple secondary antibodies can bind to a single primary antibody, increasing the fluorescence signal and enhancing sensitivity. The first step in immunofluorescence is preparing the sample. This may involve fixing the cells or tissues to preserve their structure and antigenicity. Common fixatives include formaldehyde and methanol. After fixation, permeabilization may be required to allow antibodies to penetrate cell membranes. After sample preparation, the primary antibody, specific to the target molecule of interest, is applied to the sample. The primary antibody binds to the target molecule and this step is followed by washing to remove any unbound antibodies. In indirect immunofluorescence, a secondary antibody labeled with a fluorescent dye is added to the sample. This secondary antibody recognizes and binds to the primary antibody, amplifying the signal. Again, washing is performed to remove excess secondary antibodies.

Acknowledgement

None.

Conflict of Interest

None.

References

1. Stack, Edward C., Chichung Wang, Kristin A. Roman and Clifford C. Hoyt. "Multiplexed immunohistochemistry, imaging and quantitation: A review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis."Methods70 (2014): 46-58.

Google Scholar, Crossref, Indexed at

2. Anyaegbu, Chidozie C., Tracey F. Lee-Pullen, Timothy J. Miller and Tamara N. Abel, et al. "Optimisation of multiplex immunofluorescence for a non-spectral fluorescence scanning system."J Immunol Methods 472 (2019): 25-34.

Google Scholar, Crossref, Indexed at

3. Isse, Kumiko, Andrew Lesniak, Kedar Grama and Badrinath Roysam, et al. "Digital transplantation pathology: Combining whole slide imaging, multiplex staining and automated image analysis."Am J Transplant 12 (2012): 27-37.

Google Scholar, Crossref, Indexed at

4. Hu, Wenwei, Runzi Sun, Lujun Chen and Xiao Zheng, et al. "Prognostic significance of resident CD103+ CD8+ T cells in human colorectal cancer tissues."Acta Histochem 121 (2019): 657-663.

Google Scholar, Crossref, Indexed at

5. Nicell, James A. and Harold Wright. "A model of peroxidase activity with inhibition by hydrogen peroxide."Enzyme Microb Technol 21 (1997): 302-310.

Google Scholar