Immunofluorescence Glossary

What is Immunofluorescence?

Immunofluoresence (IF) is a histochemical laboratory staining technique that relies on antibodies–antigens interactions. It uses antibodies' antigen specificity to direct fluorescent dyes to particular biomolecule targets within a cell, allowing visualization of the target molecule's distribution within the sample. IF is broadly used in clinical immunology laboratories to help in the diagnosis of autoimmune diseases and in patients' treatment and monitoring.

Introduction to Immunofluorescence

A short history of immunofluorescence and comparison to immunohistochemistry.

In 1941, Albert Hewett Coons and colleagues were able to visualize pneumococcal antigens in infected tissue specimens using fluorescently labeled antibodies. The findings were celebrated as a watershed moment, and the procedure has been endlessly developed, modified, and refined since then, resulting in an undeniably useful and flexible tool for both diagnostic and research laboratories.

Immunofluorescence can be described also as a type of IHC, wherein IF Monoclonal and polyclonal antibodies are analyzed using a fluorescence detection system, while IHC uses chemical-based approaches to detect monoclonal and polyclonal antibodies.

Similarities Between Immunofluorescence (IF) and Immunohistochemistry (IHC)

1. Both takes place under in-vitro conditions
2. Require antigen-antibody interaction
3. They have reproducible results
4. They have improved data quality
5. The techniques are rapid
6. Both can be used in diagnosis of cancers and infectious diseases

Differences Between Immunofluorescence (IF) and Immunohistochemistry (IHC)

Immunofluorescence (IF)	Immunohistochemistry (IHC )
IF is a method of identification in which fluorescent dyes or fluorescent proteins are used to mark the antibodies used in the assay.	IHC is a method of identification in which the antibodies in an assay are labelled with chemicals or radioactive elements.
IF always has a high accuracy than IHC	IHC has lower accuracy relative to IF
There is high specificity in IF	There is less specificity compared to IF

Why choose IF over IHC?

IHC is widely used to differentiate between distinct cell groups that do not overlap. IF is the staining method of choice for multiplexing, especially when co-localization is desired or when more than 2 different antibodies are needed. With IF staining, antibodies against specific proteins are added to the slide and are then visualized with fluorescent dyes and viewed with a fluorescent microscope.

Types of Immunofluorescence

There are two main types of immunofluorescence, direct and indirect immunofluorescence.

Direct Immunofluorescence

Direct IF involves use of a single antibody that is aimed towards the desired target .The fluorophore is specifically conjugated to the main antibody. The fluorophore it carries can be detected via microscopy.

Indirect Immunofluorescence

Indirect IF uses two antibodies. The primary antibody is unconjugated and a fluorophore-conjugated secondary antibody directed against the primary antibody is used for detection.

Differences Between direct and Indirect Immunofluorescence

Direct Immunofluorescence	Indirect Immunofluorescence
Relative shorter protocol, due to absence of Secondary antibody incubation.	Takes a longer time due to increased steps for Secondary antibodies.
Less background due to the absence of secondary antibodies which can cause non- specific binding.	High background due to the presence of Secondary antibodies which may result in non-specific binding.
Ability to stain with multiple antibodies at once since host factor is not a factor.	Staining with multiple antibodies can be problematic since host species needs to be considered.
Low sensitivity due to one-step staining method.	Higher sensitivity because multiple secondary antibodies can bind to one primary antibody.
Few direct antibody conjugates are readily available in the market.	Secondary antibody conjugates are readily available on the market.

Popular Terms

Below are some immunofluorescence terms and their meaning.

Immunofluorescence staining- Immunofluorescence (IF) staining uses tissue sections or cultured cell lines as an antigenic source and detects the specific recognition of autoantibodies to native autoantigens on fixed cells/tissues.

Immunofluorescence blocking- Blocking is essential for preventing non-specific binding of antibodies or other reagents to the tissue.To mitigate nonspecific binding, a blocking step should be carried out before incubation with the primary antibody.

Cyclic Immunofluorescence (CycIF) - Is a highly multiplexed method for single cell imaging. It allows fluorescent imaging to different number of proteins/signals that can be simultaneously measured.

Colocalization Immunofluorescence - Colocalization refers to the observation of the spatial overlap between two distinct fluorescent markers, each with a different emission wavelength, to see if the different "targets" are in the same region of the cell or very close to one another in fluorescence microscopy.

Double Immunofluorescence- Primary antibodies raised in different species can be used either in parallel (in a mixture) or in a sequential way to investigate the co-distribution of two (or more) distinct antigens in the same sample.

Multiplex Immunofluorescence- Immunofluorescence technologies that allow multiple markers to be detected simultaneously on a single tissue segment.

Double Immunofluorescence staining- This is a staining method of targeting two proteins within a cell or tissue with two antibodies from two different host species (eg. mouse IgG and rabbit IgG), isotypes of the same species.

FISH immunofluorescence- Fluorescence in situ hybridization (FISH) is way to visualize and map the genetic material in an individual's cells, including specific or portions of genes.

Full House immunofluorescence- This simply means that all five main immunofluorescent stains (IgM, IgG, IgA, C3, and C1q) on a renal biopsy are positive.

Linear Immunofluorescence- It is a characteristic pattern of linear immunofluorescent staining for immunoglobulin G (IgG).

Multicolor Immunofluorescence- Multicolor immunofluorescence staining is best carried out by sequentially incubating cells with unlabeled-primary and labeled-secondary antibodies. However when options are limited, it may also be performed by simultaneous incubation of cells with directly labelled primary antibodies.

Photoluminescence- the process through which atoms emit light following light absorption, includes fluorescence and phosphorescence.

Commonly Used Fluorophores;

Immunofluorescence dyes can be classified as either as Organic dyes, Biological and Inorganic Fluorophores.

Factors that affect the efficiency of fluorophores

1. Peak excitation wavelength;

2. Peak emission wavelength,

3. Quantum yield, brightness

4. Water solubility,

5. pH insensitivity;

6. photostability,

7. spillover effect

8.autofluorescence effects.

Characteristics of a good Immunofluorescence Fluorophores

1. Not alter the general shape and function of the target molecules/cells

2. Be localized at the target location on the cell/molecule

3. Maintain high specificity even in the presence of other molecules

4. Operate at visible wavelengths

Organic Dyes;

Fluorescein Dye;

Fluorescein is a highly fluorescent substance. It can be detected even when in very small quantities and is used in microscopy when it's conjugated to antibodies. It is available as a dark orange / red powder slightly soluble in water and alcohol.

Multicolor immunofluorescence staining is best carried out by sequentially incubating cells with unlabeled-primary and labeled-secondary antibodies. However when options are limited, it may also be performed by simultaneous incubation of cells with directly labelled primary antibodies. Commonly conjugated to proteins, antibodies, and nucleic acids for a variety of applications, such as histochemistry, fluorescence microscopy, FISH, and flow cytometry. The dye has an absorption wavelength that peaks around 495 nm, and an emission maximum around 517 nm.

Fluorescein Isothiocyanate (FITC)-Is a derivative of fluorescein and one of the most widely used organic fluorescent dyes/probes in flow cytometry and immunofluorescence is fluorescein isothiocyanate. With fluorescein-to-fluorescein interactions resulting in energy conversion and self-quenching while concentrated, it has a maximum/peak absorbance of 495nm and an emission wavelength of 520nm.Best reagents for biological research because of their high absorptivity, excellent fluorescence quantum yield, and good water solubility.

Cyanine Dyes-Cy3 and Cy5 are the most popular, used typically combined for 2 colors detection. Cyanines are resonant dyes characterized by polymethine dyes between nitrogen atoms (two atoms of nitrogen) with a delocalized charge so that they can be chemically linked to either nucleic acids or protein molecules. Cyanines have become one of the most common fluorescent dyes for labeling nucleic acids due to their low non-specific binding to biomolecules and bright fluorescence. Because they yield brighter and more stable fluorescence, cyanines can advantageously replace conventional dyes such as fluorescein and rhodamines.

Rhodamine -The rhodamines are xanthene derivatives structurally related to fluorescein, but with additional chemical substitutions that shift their excitation and emission spectra to longer wavelengths.Rhodamines have superior photostability and a variety of photophysical properties, making them suitable for use as laser dyes, fluorescent probes, and pigments as opposed to other dyes on the market

Pros and Cons of Organic Dyes

Pros	Cons
Photostability	A relatively high rate of photobleaching
Most are soluble in Water and Alcohol	Fluorescent signal is sensitive to pH changes
Bright Fluorescence	A relatively broad fluorescence emission spectrum
High absorptivity	Fluorescence quenching on conjugation to biopolymer
Excellent fluorescence quantum yield

Biological Fluorophores;

Green Fluorescent Protein (GFP)

Green fluorescent protein (GFP) is a protein in the jellyfish that exhibits green fluorescence when exposed to light. The protein has 238 amino acids, three of them (Numbers 65 to 67) form a structure that emits visible green fluorescent light. In the jellyfish, GFP interacts with another protein, called aequorin, which emits blue light when added with calcium. GFP is used to study cells in embryos and fetuses during developmental processes.

Applications of GFP

1. Study of bacterial protein localization

2. Host–pathogen interaction research

3. Use of GFP as reporter gene

4. GFP as active indicator

5. GFP as fusion tag

Take to me anti-GFP antibody

Phycoerythrin-PE (phycoerythrin) is a pigment complex made up of red proteins that belongs to the phycobiliprotein family. It's present in both red algae and cryptophytes, where it serves as a supplement to the chlorophyll pigments. It’s primary absorption peak is at 565 nm with secondary peaks at 496 and 545 nm. The broad excitation spectrum provides the advantage for multi-color immunofluorescent staining or cell sorting. They are significantly brighter and more photostable than conventional organic fluorophores. While it emits bright fluorescence, phycoerythrin photobleaches rather quickly, limiting its use in fluorescence microscopy.

Allophycocyanin (APC)-Allophycocyanin, like Phycoerythrin, is derived from red algae and belongs to the phycobiliprotein family. The fluorophore's absorbance limit is at 650nm, while fluorescence emission peaks at 66nm, when excited by laser lines at 594 and 633 nm. APC is fluorescent, with an extremely high absorptivity and high quantum efficiency. It is a protein which can be easily linked to antibodies and other proteins by conventional protein cross-linking techniques without altering its spectral characteristics. Allophycocyanin is the least stable among the major phycobiliproteins, susceptible to dissociation at low concentrations including concentrations at which some assays are performed. APC can be excited by light over a wide range of the visible spectrum, is highly water soluble, has a relatively low isoelectric point, and lacks potentially sticky carbohydrates

Pros and Cons of Biological Fluorophores

Pros	Cons
Relatively low isoelectric point	Photobleaches
Broad excitation spectrum	Susceptible to dissociation at low concentrations
Significantly brighter and more photostable	Toxicity
Highly Water Soluble	Interference with normal biological processes

Inorganic Fluorophores

Quantum Dots-Quantum dots are nanocrystals (inorganic nanocrystals) with sizes ranging from 2 to 50 nanometers. The light emitted varies in color from blue to red, depending on the size (with small QT exhibiting blue light while larger ones exhibit red light). They are suitable for processes such as immunolabeling, multiplexed biological detection as well as molecular imaging both in vitro and in vivo assays.They have much increased brightness, narrow emission spectrum, large Stokes shift and photostability compared with conventional organic fluorescent dyes, which together make them the fluorophores of choice for demanding requirements.

Nanoparticles-Nanoparticles can be coated/modified with fluorophores on their surfaces to make them fluorescent and improve such properties brightness, biocompatibility and cell-permeability. Compared with conventional fluorescent dyes, the use of fluorescent nanoparticles as labels in immunofluorescence microscopy offers advantages of higher luminescence and higher photostability. This method can integrate with epifluorescent filter techniques to further shorten the time needed for detection. In addition, by substituting the antibody to suit other bacteria, this technique has the potential to develop to a universal method for detecting a wide variety of bacteria in biomedical and biotechnological areas.

Pros and Cons of Inorganic Fluorophores

Pros	Cons
Have much increased brightness,	Exhibit in vivo toxicity
Narrow emission spectrum	Irregular blinking in case of Quantum dot single particle tracking.
Large Stokes shift
Higher luminescence and higher photostability

Diseases Diagnosed By Immunofluorescence

Below are some disease that are diagonised by immunofluorescence tests.

Pemphigus vulgaris- Immunofluorescence is one of the diagnostic tests done in cases of pemphigus. Pemphigus vulgaris is an autoimmune blistering condition marked by suprabasal blisters.

Antinuclear Antibody (ANA)- The antinuclear antibody (ANA) test is used as a primary test to help evaluate a person for autoimmune disorders that affect many tissues and organs throughout the body (systemic) and is most often used as one of the tests to help diagnose systemic lupus erythematosus (SLE).

Dermatitis Herpetiformis- Granular IgA deposits in dermal papillae of perilesional skin observed by direct immunofluorescence is the criterion standard of diagnosis. DH is usually confirmed with a skin biopsy and a specialized type of immunofluorescent stain that helps to detect the IgA antibodies.

Alport syndrome - Immunofluorescence analysis using the monoclonal antibody to the 28-kilodalton monomers of the non collagenous domain of type IV collagen can verify the diagnosis of heterozygous Alport syndrome.

Amyloidosis- Amyloidosis is a relatively rare disease characterized by the extracellular deposition of precursor proteins. Cardiac involvement can be especially devastating due to symptoms of diastolic dysfunction and right-sided heart failure. Diagnostic algorithm of cardiac amyloidosis of direct evaluation of the tissue with immunofluorescence and of genetic testing.

Autoimmune Bullous Dermatoses- Immunofluorescence assays plays important role in diagnosing autoimmune blistering diseases, and higher sensitivity for indirect immunofluorescence when Salt-split skin technique is performed.

Autoimmune Liver diseases- Autoimmune liver diseases comprising the triad of autoimmune hepatitis, primary biliary cholangitis (PBC) (cirrhosis) and primary sclerosing cholangitis and their overlap syndromes are uncommon. . For routine diagnostic immunology laboratories, initial screening for liver autoantibodies by immunofluorescence remains the method of choice.

Chikungunya Virus- Immunofluorescence assay (IFA) is a highly versatile and sensitive assay for detection and titration of chikungunya virus (CHIKV). The IFA technique requires virus-infected cells (viral antigen) and antibodies specific to the viral antigens for detection.

Antibodies, Proteins, and Genes associated with Immunofluorescence

Below are some of the antibodies, proteins, and genes that are mostly associated with Immunofluorescence.

Antibodies	Proteins	Genes
CD4	NRF2	EEA1
CD11b	P21	LGR5
CD3	PAX7	NFR2
CD8	PCNA	EGFR
CD31	NFKB	RAD51
CD45	LC3B	SOX2
CD68	53BP1	ALDH1A1
F4/80	C3	Tom20
LGR5	FLAG	ATPAF2
VEGF	FOXP3
2H6	Ki-67
AAV	LC3
	P16
	P53
	P62
	CXCR4