Immunofluorescence

Immunofluorescence is a technique which is used in targeting specific biomolecules present in a cell by the effective use of fluorescent dyes and a fluorescence microscope. The technique is mostly used in the analysis and study of microbiological samples. It takes advantage of the exclusive specificity of antibodies to antigen and effective visual interpretations of these interactions are made using fluorescent dyes. This aids in visualizing the exact positioning and distribution on the target molecule in question.

 Recognition sites on an antibody and antigen are a paratope and epitope respectively. An antibody binds to the epitope of its specific antigen while the antigen recognizes the paratope region in its specific antibody. The method is primarily used for immunostaining and immunohistochemistry (for tissues). In immunostaining, fluorophores are used to stain specific proteins for further study and analysis. This is especially beneficial in identification of cancerous growth, effective drug action on target proteins, role of certain proteins in cellular development etc. This method is efficacious since the fluorophore neither interferes with the antigen-antibody interaction nor affects the binding capacity of the antibody to the antigen.

The most common microscopes used for analysis are epifluorescence microscope, confocal microscope and other super resolution microscopes like stimulated emission depletion (STED) microscopy, saturated structured-illumination microscopy (SSIM), fluorescence photo-activation localization microscopy (FPALM), and stochastic optical reconstruction microscopy (STORM).

Immunofluorescence is mainly of two types: –

  1. Direct or primary immunofluorescence: It uses only a primary antibody linked to a fluorophore for recognizing and targeting an antigen. The procedure is time efficient and reduces non-specific background noise. Only a limited number of fluorophores can bind to the primary antibody, thus making the process less sensitive in comparison to its counterpart and leading to false positives. Furthermore, a massive amount of 1° antibody is required making the process all the more expensive.
  2. Indirect or secondary immunofluorescence: Two antibodies are used; one that binds to the target molecule and the other which is tagged with a fluorophore. As several secondary antibodies are capable of binding to the primary antibody, the signal is amplified to multiple accounts. The process is extremely flexible in terms of the techniques used for detection and the variations one can employ in the secondary antibodies.

Modified antibodies are also used to facilitate variation in affinity towards the target molecules. These modifications permit primary antibodies with several regions that identify a range of antigens or by introducing cross- species antibodies as primary and secondary antibodies (Eg.: Goat antibody and dye coupled rabbit antibody gives rabbit anti-goat antibodies).

Figure 1: Mode of action in primary and secondary immunofluorescence

Methodology

2.1 Fluorophore

For immunofluorescence, a fluorophore is used as a dye. It is a chemical compound which re-emits light due to excitation of light. It is, by default, a fluorescent compound. They are used by covalently bonding them to macromolecules, thus acting as a marker in the form of a dye or a tag.  These compounds aid in effective and safe staining of cells, tissues sections under observation and visualized using spectroscopy and fluorescent imaging techniques. Some examples include fluorescein isothiocyanate, carboxyfluorescein, rhodamine derivatives, coumarin, cyanine etc.

These compounds must especially show photo-stability, brightness, reduced pH sensitivity and non-reactivity. It essentially absorbs energy at a particular wavelength and re-emits it at a much longer wavelength. The electrons delocalize upon absorption of light energy, excites to another band energy level, attempts at stabilization, and returns to ground state following emission.  Some of the characteristics of fluorophore include :-

  • Its emission spectrum is a lot sharper than the excitation spectrum.
  • Molar absorption coefficient (molar -1 cm-1) which depends upon the absorbed light at a given wavelength against the concentration of the sample.
  • The quantum yield in energy transfer from incidence to emission of fluorescence.
  • The lifetime of the excited fluorophore measured in pico-seconds estimates the time from excitation to return to ground state.
  • Stokes shift calculated between maximum excitation and emission wavelengths.
Figure 2: Human epithelial intestinal and lung organoids stained with antibodies for
Acetyl-α-Tubulin (A), α-Carbonic Anhydrase IV (B) and Sox-9 (AB5535) (C).

Fluorophores are classified into:-

  1. Proteins & peptides (fluorescent proteins – GFP, YFP, RFP)
  2. Organic compounds (small)
  3. Oligomers & polymers (synthetic)
  4. Multi-component systems

Some of the non-protein organic fluorophores are classified into 13 families. Some of them include:-

  1.  Xanthene derivatives: fluorescein, eosin, and Texas red
  2. Cyanine derivatives: cyanine, thiacarbocyanine, and merocyanine 
  3. Anthracene derivatives: anthraquinones, DRAQ5, DRAQ7
  4. Oxazine derivatives: Nile redNile bluecresyl violet
  5. Acridine derivatives: proflavinacridine orangeacridine yellow
  6. Arylmethine derivatives: auraminecrystal violetmalachite green

2.2 Chemicals & Reagents

The process of immunofluorescence requires staining of the cell/tissue samples to obtain a desired visual image. There are various chemicals used during the entire process. These include:-

  1. Phosphate Buffered Saline (20X)

For making 1L of 1x PBS – Dilute about 50 ml 20x PBS to 950ml H₂O. 

 It helps in dilution, maintaining the pH and is non-toxic to cells, thus promoting effective antigen-antibody interaction and fluorescent visualization.

2. Formaldehyde (16%)

Dilute it with 1x PBS to get 4% formaldehyde. The chemical helps in the fixing the cells and retaining their shape and positioning of the proteins.

3. Blocking buffer (10ml)

It’s made by mixing 0.5 ml normal goat serum (or any antibody source of choice) with 9ml PBS and 30 μl triton X. The buffer helps in removing non-specific binding and decreases background staining.

4. Antibody Dilution Buffer (10ml)

30 μl triton X may be added to 10ml of 1x PBS and in the end 2mg of Bovine serum albumin is added.

5. Detergent

Triton X-100 is a non-ionic detergent that facilitates permeability of antibodies into the cell and respective organelles. Saponin is another detergent used against cholesterol containing membranes.


2.3 Procedure

Direct Immunofluorescence

It involves the following steps:-

  1. The sample tissue or culture cells must be prepared and their sections should be formed.
  2. Wash the sample twice using PBS.
  3. Fix the samples using 4% formaldehyde. In case if para-formaldehyde, use it in a fume hood as it is toxic.
  4. Aspirate the fixative carefully and rinse the sample twice with PBS again of about 5 minutes each.
  5. For permeability, use detergent 0.1-0.5% triton x-100 in PBS for 10 minutes. 
  6. Aspirate the detergent and rinse the samples two times again for about 5 minutes in PBS.
  7. A diluted concoction of 10% primary (1°-Ab) antibody (normal goat serum) in PBS is added for 30 minutes at room temperature.
  8. The goat serum is aspirated and incubate fluorophore-conjugated primary antibody at required dilution in PBS at 4°C overnight (ideal) or for 1 hour at 37°C.
  9. Rinse the treated sample in PBS for 5minutes in the dark.
  10.  Incubate the samples with 1 μg/ml DAPI (4’, 6-diamidino-2-phenylindole).
  11. Use a mounting medium before placing the treated samples on the fluorescent microscope for observation.

    Indirect Immunofluorescence

It includes the following steps:-

  1. The sample tissue or culture cells must be prepared and their sections should be formed.
  2. Wash the sample twice using PBS.
  3. Fix the samples using 4% formaldehyde. In case if para-formaldehyde, use it in a fume hood as it is toxic.
  4. Aspirate the fixative carefully and rinse the sample twice with PBS again of about 5 minutes each.
  5. For permeability, use detergent 0.1-0.5% triton x-100 in PBS for 10 minutes. 
  6. Aspirate the detergent and rinse the samples two times again for about 5 minutes in PBS.
  7. A diluted concoction of 10% primary (1°-Ab) antibody (normal goat serum) in PBS is added for 30 minutes at room temperature.
  8. The goat serum is aspirated and incubate fluorophore-conjugated secondary antibody at required dilution in PBS at 4°C overnight (ideal) or for 1 hour at 37°C.
  9. Rinse the treated sample in PBS for 5 minutes in the dark.
  10.  Incubate the samples with 1 μg/ml DAPI (4’, 6-diamidino-2-phenylindole). DAPI binds to those regions in the DNA right in adenine-thymine and has the ability to stain both live and dead cells.
  11.  A mounting medium is used prior to observation in the fluorescent microscope.

Applications

This techniques has miscellaneous advantages: –

  1. It is used to analyze tissue sections cell line cultures etc. for the identification and distribution of specific antibodies, disease-specific biomolecules, proteins, glycans etc.
  2. Detection of antibodies/antigens/proteins associated with an infection or disease like: immunoglobin M antibodies against Toxoplasma gondii in congenital infections; Naegleria species; enteropathogenic Escherichia coli; Mycoplasma cultures etc.
  3. Helps in visualization of structures like filaments of intermediate size and topology of cell membrane using epitopic insertion into proteins.
  4. It helps in determining the level and extent of DNA methylation and identify the localization patterns. This is also called as the ‘semi-quantitative method’.
  5. Analysis of 3-dimensional organoid structures for visualizing molecular markers participating in cellular behavior, proliferation, differentiation by whole-mount immunofluorescent staining.

Limitations

  1. Photo-bleaching: – It occurs due to high intensity of light provided over a long duration and affects the visualization of images. This can be curbed by controlling the intensity and exposure time of light, incorporating more fluorophores into sample or employ modified fluorophores that are resistant to bleaching like Alexa Fluors, Seta Fluors.
  2. Auto-fluorescence: – It occurs when the biological sample itself fluoresces and interferes with normal fluorophore activity.
  3.  Extraneous undesired specific fluorescence: – This occurs due to contamination caused by impure antigens and other contaminants.
  4.  Nonspecific fluorescence: – This leads to the loss of specificity of the probe caused by the activity of fluorophore, improper fixation using formaldehyde or even if the sample has dried out.
  5. This process can only be employed to analyze fixed or dead biological samples especially during visualization of organelles since antibodies would otherwise not penetrate the membrane while reacting with the fluorophores.  
  6. To overcome this restriction to dead cells, recombinant proteins like GFP (green fluorescent protein) and YFP (yellow fluorescent protein) can be used that naturally possess fluorescent protein domains which enable local visualization in live cells. The problem with this alternative though is that it alters the genetic makeup of the live cell.
  7. Sometimes the fixatives used may also lead to unwanted non-specific binding causing false-positive or false-negative signal generation.
  8. Many antibodies often bind to the same epitope which can also create false results and affect further analyses.

References

  1. Mandrell, R. E., Griffiss, J. M., Macher, B. A. (1988-07-01), “Lipooligosaccharides (LOS) of Neisseria gonorrhoeae and Neisseria meningitidis have components that are immunochemically similar to precursors of human blood group antigens. Carbohydrate sequence specificity of the mouse monoclonal antibodies that recognize crossreacting antigens on LOS and human erythrocytes”Journal of Experimental Medicine168 (1): 107–126. doi:10.1084/jem.168.1.107ISSN 0022-1007PMC 2188965PMID 2456365
  2. Akiyoshi, K. (1983-01-01). Immunofluorescence in medical science: with 28 tab. Springer u.aISBN 978-3540124832OCLC 643714056
  3. Leung, Bonnie, O., Chou, K.C. (2011-09-01). “Review of Super-Resolution Fluorescence Microscopy for Biology”. Applied Spectroscopy65 (9): 967–980. doi:10.1366/11-06398ISSN 0003-7028PMID 21929850
  4.  “Immunohistochemical Staining Methods” (PDF). IHC Guidebook (Sixth ed.). Dako Denmark A/S, An Agilent Technologies Company. 2013.
  5. Juan, C.S., Alfonso B.C. (2017). “Chapter 3 Dyes and Fluorochromes”Fluorescence Microscopy in Life Sciences. Bentham Science Publishers. pp. 61–95. ISBN 978-1-68108-519-7. Retrieved 24 December 2017
  6. Rietdorf, J. (2005). Microscopic Techniques. Advances in Biochemical Engineering / Biotechnology. Berlin: Springer. pp. 246–9. ISBN 3-540-23698-8. Retrieved 2008-12-13.
  7. Tsien, R.Y., Waggoner, A. (1995). “Fluorophores for confocal microscopy”. In Pawley JB (ed.). Handbook of biological confocal microscopy. New York: Plenum Press. pp. 267–74. ISBN 0-306-44826-2. Retrieved 2008-12-13.
  8. Retrieved from the World Wide Web: https://en.wikipedia.org/wiki/Immunofluorescence

Published by Allena Andress

||Bibliophile|| Biotechnologist|| Aspiring Forensic Science specialist|| Researcher|| Closet singer and dancer|| Motivational Speaker||

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