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==Introduction==

==Introduction==

[[Image:763px-Cryptosporidium parvum 01.jpg|thumb|right|150px|Immunofluorescence of ''Cryptosporidium parvum'' spores]]

[[Image:763px-Cryptosporidium parvum 01.jpg|thumb|right|150px|Immunofluorescence of ''Cryptosporidium parvum'' spores, EPA/H.D.A. Lindquist, '''WikiMedia Commons''', 2006]]

Immunofluorescence is a technique used to detect cell or tissue-associated antigens using antibodies labelled with fluorescent tags. The stained tissues are then detected by immunofluorescence microscopy (qualitative) or flow cytometry (quantitative). Antibodies bind stably and specifically to their corresponding antigen and the technique makes use of the fact that they can be coupled to fluorescent dyes, such as fluorescein and rhodamine, with no effect on specificity. These conjugates bind to antigens present in a sample and can then be visualised under a microscope with a suitable light source, such as UV light.

Immunofluorescence is a technique used to detect cell or tissue-associated antigens using antibodies labeled with fluorescent tags. The stained tissues are then detected by immunofluorescence microscopy (qualitative) or flow cytometry (quantitative). Antibodies bind stably and specifically to their corresponding antigen and the technique makes use of the fact that they can be coupled to fluorescent dyes, such as fluorescein and rhodamine, with no effect on specificity. These conjugates bind to antigens present in a sample and can then be visualised under a microscope with a suitable light source, such as UV light.

==Fluorescent dyes==

==Fluorescent dyes==

 

If a molecule has the property of fluorescence, it can absorb light of one wavelength (excitation) and emit light of another (emission). Antibodies tagged with these dyes (known as '''fluorochromes''') form immune complexes with specific antigens which can then be indirectly visualised when excited by light of the appropriate wavelength.  

If a molecule has the property of fluorescence, it can absorb light of one wavelength (excititation) and emit light of another (emission). Antibodies tagged with these dyes (known as '''fluorochromes''') form immune complexes with specific antigens which can then be indirectly visualised when excited by light of the appropriate wavelength.  

===Commonly used fluorochromes===

===Commonly used fluorochromes===

*An antibody directed against a specific antigen is directly conjugated with the fluorescent dye and applied to the sample.  

*An antibody directed against a specific antigen is directly conjugated with the fluorescent dye and applied to the sample.  

'''Indirect staining'''

'''Indirect staining'''

*Utilizes a double layer technique- a primary, unlabelled antibody is applied to the sample, followed by a secondary antibody, an anti-immunoglobulin that has been conjugated to a fluorochrome.  

*Utilizes a double layer technique - a primary, unlabelled antibody is applied to the sample, followed by a secondary antibody, an anti-immunoglobulin that has been conjugated to a fluorochrome.  

**Indirect staining has several advantages:

**Indirect staining has several advantages:

***Several secondary antibodies bind to each primary antibody, so the resulting fluorescence is brighter than that of the direct staining.

***Several secondary antibodies bind to each primary antibody, so the resulting fluorescence is brighter than that of the direct staining.

***One preparation of secondary antibody can be used to test many sera

***One preparation of secondary antibody can be used to test many sera

***By using a mixture of primary antibodies, it is possible to detect the relative expressions of different antigens in the same cell

***By using a mixture of primary antibodies, it is possible to detect the relative expressions of different antigens in the same cell

***Quite often loss of antibody is sustained during the conjugation- in the indirect method the primary antibodies do not need to be conjugated, so this limiting factor is reduced.  

***Quite often loss of antibody is sustained during the conjugation- in the indirect method the primary antibodies do not need to be conjugated, so this limiting factor is reduced.

==Applications==

==Applications==

*Immunofluorescence is often used to identify populations of cells, and was successfully employed in the identification of the CD4+ and CD8+ subpopulations of T cells

*Immunofluorescence is often used to identify populations of cells, and was successfully employed in the identification of the CD4+ and CD8+ subpopulations of T cells

*Identifying bacterial species

*Identifying bacterial and viral species

*Detecting antigen-antibody complexes in autoimmune diseases

*Detecting antigen-antibody complexes in autoimmune diseases

*Detecting complement components in tissues

*Detecting complement components in tissues

===Flow cytometry===

===Flow cytometry===

*The flow cytometer is an instrument that directs cells in single file through a narrow chamber illuminated by a laser beam. As fluorescently-tagged cells pass through the laser beam, the fluorochrome is excited and emits light that is detected by a PMT-like detector.

*The flow cytometer is an instrument that directs cells in single file through a narrow chamber illuminated by a laser beam. As fluorescently-tagged cells pass through the laser beam, the fluorochrome is excited and emits light that is detected by a PMT-like detector

*This detector is capable of measuring the strength of emission, so it is possible to sort cells that are negative, slightly positive and highly positive for a specific antigen.

*This detector is capable of measuring the strength of emission, so it is possible to sort cells that are negative, slightly positive and highly positive for a specific antigen