Difference between revisions of "Immunofluorescence"
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==Introduction== | ==Introduction== | ||
| − | [[Image:763px-Cryptosporidium parvum 01.jpg|thumb|right|150px|Immunofluorescence of | + | [[Image:763px-Cryptosporidium parvum 01.jpg|thumb|right|150px|Immunofluorescence of Cryptosporidium parvum spores]] |
| − | Immunofluorescence is a technique used to detect cell or tissue-associated antigens using antibodies | + | 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. Conversely, the technique can also be used to detect antibodies directed against antigens known to exist in a sample. |
==Fluorescent dyes== | ==Fluorescent dyes== | ||
| − | If a molecule has the property of fluorescence, it can absorb light of one wavelength ( | + | |
| + | If a molecule has the property of fluorescence, it can absorb light of a one wavelength (excititation) and emit light of another (emission). Antibodies tagged with these dyes (known as '''fluorochromes''') form immune complexes with specific antigens that can be detected when excited by light of a certain wavelength. | ||
===Commonly used fluorochromes=== | ===Commonly used fluorochromes=== | ||
| − | *'''Fluorescein:''' organic (carbon-based) dye, most widely used. Absorbs blue (490nm) and emits yellow green fluorescence (517nm) | + | *'''Fluorescein:''' organic (carbon-based) dye, most widely used. Absorbs blue (490nm) and emits yellow green fluorescence(517nm) |
*'''Rhodamine:''' organic dye, absorbs yellow-green light and emits deep red fluorescence (546nm). | *'''Rhodamine:''' organic dye, absorbs yellow-green light and emits deep red fluorescence (546nm). | ||
**As fluorescein and rhodamine fluorescences are easy to distinguish from one another, it is possible to conjugate them to different antibodies and simultaneously visualise two different antigens on the same cell or tissue. | **As fluorescein and rhodamine fluorescences are easy to distinguish from one another, it is possible to conjugate them to different antibodies and simultaneously visualise two different antigens on the same cell or tissue. | ||
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*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 | + | *Identifying bacterial 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 | ||
| − | *Localising hormones, antigens and other cellular products in tissue | + | *Localising hormones, antigens and other cellular products in tissue sampler and subcellular compartments |
*Mapping the molecular architecture of tissues in relation to gross anatomy | *Mapping the molecular architecture of tissues in relation to gross anatomy | ||
==Developments== | ==Developments== | ||
===Confocal microscopy=== | ===Confocal microscopy=== | ||
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Revision as of 15:06, 22 August 2008
Introduction
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. Conversely, the technique can also be used to detect antibodies directed against antigens known to exist in a sample.
Fluorescent dyes
If a molecule has the property of fluorescence, it can absorb light of a one wavelength (excititation) and emit light of another (emission). Antibodies tagged with these dyes (known as fluorochromes) form immune complexes with specific antigens that can be detected when excited by light of a certain wavelength.
Commonly used fluorochromes
- Fluorescein: organic (carbon-based) dye, most widely used. Absorbs blue (490nm) and emits yellow green fluorescence(517nm)
- Rhodamine: organic dye, absorbs yellow-green light and emits deep red fluorescence (546nm).
- As fluorescein and rhodamine fluorescences are easy to distinguish from one another, it is possible to conjugate them to different antibodies and simultaneously visualise two different antigens on the same cell or tissue.
- Phycoerythrin: can absorb light from the blue-green (495nm) and the yellow wavelengths, emits bright red fluorescence
Techniques
Direct staining
- An antibody directed against a specific antigen is directly conjugated with the fluorescent dye and applied to the sample.
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.
- 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.
- 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
- 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.
- Indirect staining has several advantages:
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
- Identifying bacterial species
- Detecting antigen-antibody complexes in autoimmune diseases
- Detecting complement components in tissues
- Localising hormones, antigens and other cellular products in tissue sampler and subcellular compartments
- Mapping the molecular architecture of tissues in relation to gross anatomy