Immunofluorescence

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Vasculature of porcine skin under fluorescence (Smooth muscle actin with AlexaFluor 488). Green = smooth muscle actin (SMA) with Alexa 488 fluorophore. Blue = DAPI counterstain. Red = auto-fluorescence. Blood vessels in porcine skin - SMA A488 - 20x.jpg
Vasculature of porcine skin under fluorescence (Smooth muscle actin with AlexaFluor 488). Green = smooth muscle actin (SMA) with Alexa 488 fluorophore. Blue = DAPI counterstain. Red = auto-fluorescence.

Immunofluorescence(IF) is a light microscopy-based technique that allows detection and localization of a wide variety of target biomolecules within a cell or tissue at a quantitative level. The technique utilizes the binding specificity of antibodies and antigens. [1] The specific region an antibody recognizes on an antigen is called an epitope. Several antibodies can recognize the same epitope but differ in their binding affinity. The antibody with the higher affinity for a specific epitope will surpass antibodies with a lower affinity for the same epitope. [2] [3]

Contents

By conjugating the antibody to a fluorophore, the position of the target biomolecule is visualized by exciting the fluorophore and measuring the emission of light in a specific predefined wavelength using a fluorescence microscope. It is imperative that the binding of the fluorophore to the antibody itself, do not interfere with the immunological specificity of the antibody or the binding capacity of its antigen. [4] [5]

Immunofluorescence is a widely used example of immunostaining (using antibodies to stain proteins) and is a specific example of immunohistochemistry (the use of the antibody-antigen relationship in tissues). This technique primarily utilizes fluorophores to visualize the location of the antibodies, while others provoke a color change in the environment containing the antigen of interest or make use of a radioactive label. Immunofluorescent techniques that utilized labelled antibodies was conceptualized in the 1940s by Albert H. Coons. [2] [6] [7]

Photomicrograph of a histological section of human skin prepared for direct immunofluorescence using an anti-IgG antibody. The skin is from a patient with systemic lupus erythematosus and shows IgG deposit at two different places: The first is a band-like deposit along the epidermal basement membrane ("lupus band test" is positive). The second is within the nuclei of the epidermal cells (anti-nuclear antibodies). Lupus band test.jpg
Photomicrograph of a histological section of human skin prepared for direct immunofluorescence using an anti-IgG antibody. The skin is from a patient with systemic lupus erythematosus and shows IgG deposit at two different places: The first is a band-like deposit along the epidermal basement membrane ("lupus band test" is positive). The second is within the nuclei of the epidermal cells (anti-nuclear antibodies).

Immunofluorescence is employed in foundational scientific investigations and clinical diagnostic endeavors, showcasing its multifaceted utility across diverse substrates, including tissue sections, cultured cell lines, or individual cells. Its usage includes analysis of the distribution of proteins, glycans, small biological and non-biological molecules, and visualization of structures such as intermediate-sized filaments. [8]

If the topology of a cell membrane is undetermined, epitope insertion into proteins can be used in conjunction with immunofluorescence to determine structures within the cell membrane. [9] Immunofluorescence (IF) can also be used as a “semi-quantitative” method to gain insight into the levels and localization patterns of DNA methylation. IF can additionally be used in combination with other, non-antibody methods of fluorescent staining, e.g., the use of DAPI to label DNA. [10] [11]

Examination of immunofluorescence specimens can be conducted utilizing various microscope configurations, including the epifluorescence microscope, confocal microscope, and widefield microscope. [12]

Types

Main antinuclear antibody patterns on immunofluorescence. Main antinuclear antibody patterns on immunofluorescence.png
Main antinuclear antibody patterns on immunofluorescence.

Preparation of fluorescence

To perform immunofluorescence staining, a fluorophore must be conjugated (“tagged”) to an antibody. Staining procedures can be applied to both retained intracellular expressed antibodies, or to cell surface antigens on living cells. There are two general classes of immunofluorescence techniques: primary (direct) and secondary (indirect). [1] [2] The following descriptions will focus primarily on these classes in terms of conjugated antibodies. [12]

Primary (direct)

Basic concept of Primary Immunofluorescence: An antibody with a conjugated fluorophore, that is specifically bound to an epitope on the target molecule. Primary Immunofluorescence.png
Basic concept of Primary Immunofluorescence: An antibody with a conjugated fluorophore, that is specifically bound to an epitope on the target molecule.

Primary (direct) immunofluorescence (DIF) uses a single antibody, conjugated to a fluorophore. The antibody recognizes the target molecule (antigen) and binds to a specific region, called the epitope. The attached fluorophore can be detected via fluorescent microscopy, which, depending on the type of fluorophore, will emit a specific wavelength of light once excited. [1] [14]

The direct attachment of the fluorophore to the antibody reduces the number of steps in the sample preparation procedure, saving time and reducing non-specific background signal during analysis. [12] This also limits the possibility of antibody cross-reactivity, and possible mistakes throughout the process. One disadvantage of DIF is the limited number of antibodies that can bind to the antigen. This limitation may reduce sensitivity to the technique. When the target protein is available in only small concentrations, a better approach would be secondary IF, which is considered to be more sensitive than DIF [2] [12] when compared to Secondary (Indirect) Immunofluorescence. [1]

Basic concept of Secondary Immunofluorescence: Secondary antibody, with a conjugated fluorophore, bound to a primary antibody that is specifically bound to an epitope on the target molecule. Secondary Immunofluorescence.png
Basic concept of Secondary Immunofluorescence: Secondary antibody, with a conjugated fluorophore, bound to a primary antibody that is specifically bound to an epitope on the target molecule.

Secondary (indirect)

Secondary (indirect) immunofluorescence (SIF) is similar to direct immunofluorescence, however the technique utilizes two types of antibodies whereas only one of them have a conjugated fluorophore. The antibody with the conjugated fluorophore is referred to as the secondary antibody, while the unconjugated is referred to as the primary antibody. [1]

The principle of this technique is that the primary antibody specifically binds to the epitope on the target molecule, whereas the secondary antibody, with the conjugated fluorophore, recognizes and binds to the primary antibody. [1]  

This technique is considered to be more sensitive than primary immunofluorescence, because multiple secondary antibodies can bind to the same primary antibody. The increased number of fluorophore molecules per antigen increases the amount of emitted light, and thus amplifies the signal. [1] There are different methods for attaining a higher fluorophore-antigen ratio such as the Avidin-Biotin Complex (ABC method) and Labeled Streptavidin-Biotin (LSAB method). [15] [16]

Limitations

Basic concept of the ABC-method: Primary antibody binds to the antigen, before binding to the biotinylated secondary antibody. Avidin-Biotin enzyme complex (ABC) then attaches to the secondary antibody. ABC-method.png
Basic concept of the ABC-method: Primary antibody binds to the antigen, before binding to the biotinylated secondary antibody. Avidin-Biotin enzyme complex (ABC) then attaches to the secondary antibody.

Immunofluorescence is only limited to fixed (i.e. dead) cells, when studying structures within the cell, as antibodies generally do not penetrate intact cellular or subcellular membranes in living cells, because they are large proteins. To visualize these structures, antigenic material must be fixed firmly on its natural localization inside the cell. [17] To study structures within living cells, in combination with fluorescence, one can utilize recombinant proteins containing fluorescent protein domains, e.g., green fluorescent protein (GFP). The GFP-technique involves altering the genetic information of the cells. [18] [19]

A significant problem with immunofluorescence is photobleaching, [12] the fluorophores permanent loss of ability to emit light. [1] To mitigate the risk of photobleaching one can employ different strategies. By reducing or limiting the intensity, or timespan of light exposure, the absorption-emission cycle of fluorescent light is decreased, thus preserving the fluorophores functionality. One can also increase the concentration of fluorophores, or opt for more robust fluorophores that exhibit resilience against photobleaching such as Alexa Fluors, Seta Fluors, or DyLight Fluors. [2]

Basic concept of the LSAB-method: Utilizes a Streptavidin-enzyme conjugate for the identification of the biotinylated secondary antibody which is bound to the primary antibody. This approach is applicable when the Avidin-Biotin complex in the ABC method becomes too large. LSAB-method.jpg
Basic concept of the LSAB-method: Utilizes a Streptavidin–enzyme conjugate for the identification of the biotinylated secondary antibody which is bound to the primary antibody. This approach is applicable when the Avidin–Biotin complex in the ABC method becomes too large.

Other problems that may arise when using immunofluorescence techniques include autofluorescence, spectral overlap and non-specific staining. [1] [2] Autofluorescence includes the natural fluorescence emitted from the sample tissue or cell itself. Spectral overlap happens when a fluorophore has a broad emission specter, that overlaps with the specter of another fluorophore, thus giving rise to false signals. Non-specific staining occurs when the antibody, containing the fluorophore, binds to unintended proteins because of sufficient similarity in the epitope. This can lead to false positives. [2] [4] [1]

Advances

The main improvements to immunofluorescence lie in the development of fluorophores and fluorescent microscopes. Fluorophores can be structurally modified to improve brightness and photostability, while preserving spectral properties and cell permeability. [20]

Photomicrograph of a histological section of human skin prepared for direct immunofluorescence using an anti-IgA antibody. The skin is from a patient with Henoch-Schonlein purpura: IgA deposits are found in the walls of small superficial capillaries (yellow arrows). The pale wavy green area on top is the epidermis, the bottom fibrous area is the dermis. HSP IF IgA.jpg
Photomicrograph of a histological section of human skin prepared for direct immunofluorescence using an anti-IgA antibody. The skin is from a patient with Henoch–Schönlein purpura: IgA deposits are found in the walls of small superficial capillaries (yellow arrows). The pale wavy green area on top is the epidermis, the bottom fibrous area is the dermis.

Super-resolution fluorescence microscopy methods can produce images with a higher resolution than those microscopes imposed by the diffraction limit. This enables the determination of structural details within the cell. [21] Super-resolution in fluorescence, more specifically, refers to the ability of a microscope to prevent the simultaneous fluorescence of adjacent spectrally identical fluorophores (spectral overlap). Some of the recently developed super-resolution fluorescent microscope methods include stimulated emission depletion (STED) microscopy, saturated structured-illumination microscopy (SSIM), fluorescence photoactivation localization microscopy (FPALM), and stochastic optical reconstruction microscopy (STORM). [22]

Notable people

See also

Related Research Articles

<span class="mw-page-title-main">Immunostaining</span> Biochemical technique

In biochemistry, immunostaining is any use of an antibody-based method to detect a specific protein in a sample. The term "immunostaining" was originally used to refer to the immunohistochemical staining of tissue sections, as first described by Albert Coons in 1941. However, immunostaining now encompasses a broad range of techniques used in histology, cell biology, and molecular biology that use antibody-based staining methods.

<span class="mw-page-title-main">Fluorophore</span> Agents that emit light after excitation by light

A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with several π bonds.

<span class="mw-page-title-main">Immunohistochemistry</span> Common application of immunostaining

Immunohistochemistry (IHC) is a form of immunostaining. It involves the process of selectively identifying antigens (proteins) in cells and tissue, by exploiting the principle of antibodies binding specifically to antigens in biological tissues. Albert Hewett Coons, Ernest Berliner, Norman Jones and Hugh J Creech was the first to develop immunofluorescence in 1941. This led to the later development of immunohistochemistry.

<span class="mw-page-title-main">Fluorescence microscope</span> Optical microscope that uses fluorescence and phosphorescence

A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image.

<span class="mw-page-title-main">Direct fluorescent antibody</span>

A direct fluorescent antibody, also known as "direct immunofluorescence", is an antibody that has been tagged in a direct fluorescent antibody test. Its name derives from the fact that it directly tests the presence of an antigen with the tagged antibody, unlike western blotting, which uses an indirect method of detection, where the primary antibody binds the target antigen, with a secondary antibody directed against the primary, and a tag attached to the secondary antibody.

<span class="mw-page-title-main">Single-domain antibody</span> Antibody fragment

A single-domain antibody (sdAb), also known as a Nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. With a molecular weight of only 12–15 kDa, single-domain antibodies are much smaller than common antibodies which are composed of two heavy protein chains and two light chains, and even smaller than Fab fragments and single-chain variable fragments.

<span class="mw-page-title-main">Texas Red</span> Chemical compound

Texas Red or sulforhodamine 101 acid chloride is a red fluorescent dye, used in histology for staining cell specimens, for sorting cells with fluorescent-activated cell sorting machines, in fluorescence microscopy applications, and in immunohistochemistry. Texas Red fluoresces at about 615 nm, and the peak of its absorption spectrum is at 589 nm. The powder is dark purple. Solutions can be excited by a dye laser tuned to 595-605 nm, or less efficiently a krypton laser at 567 nm. The absorption extinction coefficient at 596 nm is about 85,000 M−1cm−1.

<span class="mw-page-title-main">Photobleaching</span> Loss of colour by a pigment when illuminated

In optics, photobleaching is the photochemical alteration of a dye or a fluorophore molecule such that it is permanently unable to fluoresce. This is caused by cleaving of covalent bonds or non-specific reactions between the fluorophore and surrounding molecules. Such irreversible modifications in covalent bonds are caused by transition from a singlet state to the triplet state of the fluorophores. The number of excitation cycles to achieve full bleaching varies. In microscopy, photobleaching may complicate the observation of fluorescent molecules, since they will eventually be destroyed by the light exposure necessary to stimulate them into fluorescing. This is especially problematic in time-lapse microscopy.

<span class="mw-page-title-main">STED microscopy</span> Technique in fluorescence microscopy

Stimulated emission depletion (STED) microscopy is one of the techniques that make up super-resolution microscopy. It creates super-resolution images by the selective deactivation of fluorophores, minimizing the area of illumination at the focal point, and thus enhancing the achievable resolution for a given system. It was developed by Stefan W. Hell and Jan Wichmann in 1994, and was first experimentally demonstrated by Hell and Thomas Klar in 1999. Hell was awarded the Nobel Prize in Chemistry in 2014 for its development. In 1986, V.A. Okhonin had patented the STED idea. This patent was unknown to Hell and Wichmann in 1994.

<span class="mw-page-title-main">Immunocytochemistry</span>

Immunocytochemistry (ICC) is a common laboratory technique that is used to anatomically visualize the localization of a specific protein or antigen in cells by use of a specific primary antibody that binds to it. The primary antibody allows visualization of the protein under a fluorescence microscope when it is bound by a secondary antibody that has a conjugated fluorophore. ICC allows researchers to evaluate whether or not cells in a particular sample express the antigen in question. In cases where an immunopositive signal is found, ICC also allows researchers to determine which sub-cellular compartments are expressing the antigen.

Fluorescence Loss in Photobleaching (FLIP) is a fluorescence microscopy technique used to examine movement of molecules inside cells and membranes. A cell membrane is typically labeled with a fluorescent dye to allow for observation. A specific area of this labeled section is then bleached several times using the beam of a confocal laser scanning microscope. After each imaging scan, bleaching occurs again. This occurs several times, to ensure that all accessible fluorophores are bleached since unbleached fluorophores are exchanged for bleached fluorophores, causing movement through the cell membrane. The amount of fluorescence from that region is then measured over a period of time to determine the results of the photobleaching on the cell as a whole.

<span class="mw-page-title-main">Immunolabeling</span> Procedure for detection and localization of an antigen

Immunolabeling is a biochemical process that enables the detection and localization of an antigen to a particular site within a cell, tissue, or organ. Antigens are organic molecules, usually proteins, capable of binding to an antibody. These antigens can be visualized using a combination of antigen-specific antibody as well as a means of detection, called a tag, that is covalently linked to the antibody. If the immunolabeling process is meant to reveal information about a cell or its substructures, the process is called immunocytochemistry. Immunolabeling of larger structures is called immunohistochemistry.

<span class="mw-page-title-main">Fluorescence in the life sciences</span> Scientific investigative technique

Fluorescence is used in the life sciences generally as a non-destructive way of tracking or analysing biological molecules. Some proteins or small molecules in cells are naturally fluorescent, which is called intrinsic fluorescence or autofluorescence. Alternatively, specific or general proteins, nucleic acids, lipids or small molecules can be "labelled" with an extrinsic fluorophore, a fluorescent dye which can be a small molecule, protein or quantum dot. Several techniques exist to exploit additional properties of fluorophores, such as fluorescence resonance energy transfer, where the energy is passed non-radiatively to a particular neighbouring dye, allowing proximity or protein activation to be detected; another is the change in properties, such as intensity, of certain dyes depending on their environment allowing their use in structural studies.

Chromogenic in situ hybridization (CISH) is a cytogenetic technique that combines the chromogenic signal detection method of immunohistochemistry (IHC) techniques with in situ hybridization. It was developed around the year 2000 as an alternative to fluorescence in situ hybridization (FISH) for detection of HER-2/neu oncogene amplification. CISH is similar to FISH in that they are both in situ hybridization techniques used to detect the presence or absence of specific regions of DNA. However, CISH is much more practical in diagnostic laboratories because it uses bright-field microscopes rather than the more expensive and complicated fluorescence microscopes used in FISH.

Super-resolution microscopy is a series of techniques in optical microscopy that allow such images to have resolutions higher than those imposed by the diffraction limit, which is due to the diffraction of light. Super-resolution imaging techniques rely on the near-field or on the far-field. Among techniques that rely on the latter are those that improve the resolution only modestly beyond the diffraction-limit, such as confocal microscopy with closed pinhole or aided by computational methods such as deconvolution or detector-based pixel reassignment, the 4Pi microscope, and structured-illumination microscopy technologies such as SIM and SMI.

<span class="mw-page-title-main">Immunogold labelling</span> Staining technique used in electron microscopy

Immunogold labeling or immunogold staining (IGS) is a staining technique used in electron microscopy. This staining technique is an equivalent of the indirect immunofluorescence technique for visible light. Colloidal gold particles are most often attached to secondary antibodies which are in turn attached to primary antibodies designed to bind a specific antigen or other cell component. Gold is used for its high electron density which increases electron scatter to give high contrast 'dark spots'.

<span class="mw-page-title-main">Chromatin bridge</span> Medical condition

Chromatin bridge is a mitotic occurrence that forms when telomeres of sister chromatids fuse together and fail to completely segregate into their respective daughter cells. Because this event is most prevalent during anaphase, the term anaphase bridge is often used as a substitute. After the formation of individual daughter cells, the DNA bridge connecting homologous chromosomes remains fixed. As the daughter cells exit mitosis and re-enter interphase, the chromatin bridge becomes known as an interphase bridge. These phenomena are usually visualized using the laboratory techniques of staining and fluorescence microscopy.

Photo-activated localization microscopy and stochastic optical reconstruction microscopy (STORM) are widefield fluorescence microscopy imaging methods that allow obtaining images with a resolution beyond the diffraction limit. The methods were proposed in 2006 in the wake of a general emergence of optical super-resolution microscopy methods, and were featured as Methods of the Year for 2008 by the Nature Methods journal. The development of PALM as a targeted biophysical imaging method was largely prompted by the discovery of new species and the engineering of mutants of fluorescent proteins displaying a controllable photochromism, such as photo-activatible GFP. However, the concomitant development of STORM, sharing the same fundamental principle, originally made use of paired cyanine dyes. One molecule of the pair, when excited near its absorption maximum, serves to reactivate the other molecule to the fluorescent state.

The enzyme-linked immunosorbent spot (ELISpot) is a type of assay that focuses on quantitatively measuring the frequency of cytokine secretion for a single cell. The ELISpot Assay is also a form of immunostaining since it is classified as a technique that uses antibodies to detect a protein analyte, with the word analyte referring to any biological or chemical substance being identified or measured.

<span class="mw-page-title-main">Fluorescence imaging</span> Type of non-invasive imaging technique

Fluorescence imaging is a type of non-invasive imaging technique that can help visualize biological processes taking place in a living organism. Images can be produced from a variety of methods including: microscopy, imaging probes, and spectroscopy.

References

  1. 1 2 3 4 5 6 7 8 9 10 Odell ID, Cook D (2013-01-01). "Immunofluorescence Techniques". Journal of Investigative Dermatology. 133 (1): e4. doi: 10.1038/jid.2012.455 . PMID   23299451.
  2. 1 2 3 4 5 6 7 Joshi S, Yu D (2017), "Immunofluorescence", Basic Science Methods for Clinical Researchers, Elsevier, pp. 135–150, doi:10.1016/b978-0-12-803077-6.00008-4, ISBN   978-0-12-803077-6 , retrieved 2024-02-14
  3. ↑ Ladner RC (2007-01-01). "Mapping the epitopes of antibodies". Biotechnology & Genetic Engineering Reviews. 24 (1): 1–30. CiteSeerX   10.1.1.536.6172 . doi:10.1080/02648725.2007.10648092. PMID   18059626. S2CID   34595289.
  4. 1 2 Marks KM, Nolan GP (August 2006). "Chemical labeling strategies for cell biology". Nature Methods. 3 (8): 591–596. doi:10.1038/nmeth906. ISSN   1548-7091. PMID   16862131. S2CID   27848267.
  5. ↑ Owenius R, Österlund M, Lindgren M, Svensson M, Olsen OH, Persson E, Freskgård PO, Carlsson U (October 1999). "Properties of Spin and Fluorescent Labels at a Receptor-Ligand Interface". Biophysical Journal. 77 (4): 2237–2250. Bibcode:1999BpJ....77.2237O. doi:10.1016/S0006-3495(99)77064-5. PMC   1300504 . PMID   10512843.
  6. ↑ Hökfelt T (November 1999). "Neurobiology thanks to microbiology: The legacy of Albert H. Coons (1912–1978)". Brain Research Bulletin. 50 (5–6): 371–372. doi:10.1016/S0361-9230(99)00109-4. PMID   10643440. S2CID   33618171.
  7. ↑ Sheng W, Zhang C, Mohiuddin TM, Al-Rawe M, Zeppernick F, Falcone FH, Meinhold-Heerlein I, Hussain AF (2023-02-04). "Multiplex Immunofluorescence: A Powerful Tool in Cancer Immunotherapy". International Journal of Molecular Sciences. 24 (4): 3086. doi: 10.3390/ijms24043086 . ISSN   1422-0067. PMC   9959383 . PMID   36834500.
  8. ↑ Franke WW, Schmid E, Osborn M, Weber K (October 1978). "Different intermediate-sized filaments distinguished by immunofluorescence microscopy". Proceedings of the National Academy of Sciences of the United States of America. 75 (10): 5034–5038. Bibcode:1978PNAS...75.5034F. doi: 10.1073/pnas.75.10.5034 . PMC   336257 . PMID   368806.
  9. ↑ Wang H, Lee EW, Cai X, Ni Z, Zhou L, Mao Q (December 2008). "Membrane topology of the human breast cancer resistance protein (BCRP/ABCG2) determined by epitope insertion and immunofluorescence". Biochemistry. 47 (52): 13778–13787. doi:10.1021/bi801644v. PMC   2649121 . PMID   19063604.
  10. ↑ Çelik S (January 2015). "Understanding the complexity of antigen retrieval of DNA methylation for immunofluorescence-based measurement and an approach to challenge". Journal of Immunological Methods. 416: 1–16. doi:10.1016/j.jim.2014.11.011. PMID   25435341.
  11. ↑ Grimason A, Smith H, Parker J, Bukhari Z, Campbell A, Robertson L (March 1994). "Application of DAPI and immunofluorescence for enhanced identification of Cryptosporidium spp oocysts in water samples". Water Research. 28 (3): 733–736. Bibcode:1994WatRe..28..733G. doi:10.1016/0043-1354(94)90154-6.
  12. 1 2 3 4 5 Piña R, Santos-Díaz AI, Orta-Salazar E, Aguilar-Vazquez AR, Mantellero CA, Acosta-Galeana I, Estrada-Mondragon A, Prior-Gonzalez M, Martinez-Cruz JI, Rosas-Arellano A (2022-01-26). "Ten Approaches That Improve Immunostaining: A Review of the Latest Advances for the Optimization of Immunofluorescence". International Journal of Molecular Sciences. 23 (3): 1426. doi: 10.3390/ijms23031426 . ISSN   1422-0067. PMC   8836139 . PMID   35163349.
  13. ↑ Al-Mughales JA (2022). "Anti-Nuclear Antibodies Patterns in Patients With Systemic Lupus Erythematosus and Their Correlation With Other Diagnostic Immunological Parameters". Front Immunol. 13: 850759. doi: 10.3389/fimmu.2022.850759 . PMC   8964090 . PMID   35359932.
    Minor edits by Mikael Häggström, MD
    - Attribution 4.0 International (CC BY 4.0) license
  14. ↑ "Immunohistochemical Staining Methods" (PDF). IHC Guidebook (Sixth ed.). Dako Denmark A/S, An Agilent Technologies Company. 2013. Archived from the original (PDF) on 2016-08-03. Retrieved 2014-05-14.
  15. ↑ Im K, Mareninov S, Diaz MF, Yong WH (2019), Yong WH (ed.), "An Introduction to Performing Immunofluorescence Staining", Biobanking, vol. 1897, New York, NY: Springer New York, pp. 299–311, doi:10.1007/978-1-4939-8935-5_26, ISBN   978-1-4939-8933-1, PMC   6918834 , PMID   30539454
  16. ↑ Yarilin D, Xu K, Turkekul M, Fan N, Romin Y, Fijisawa S, Barlas A, Manova-Todorova K (2015-03-31). "Machine-based method for multiplex in situ molecular characterization of tissues by immunofluorescence detection". Scientific Reports. 5 (1): 9534. Bibcode:2015NatSR...5E9534Y. doi:10.1038/srep09534. ISSN   2045-2322. PMC   4821037 . PMID   25826597.
  17. ↑ "Fixation and Permeabilization in in[sic] Immunocytochemistry/Immunofluorescence (ICC/IF)". Novus Biologicals. 2024-02-14. Retrieved 2024-02-14.
  18. ↑ Ehrhardt D (December 2003). "GFP technology for live cell imaging". Current Opinion in Plant Biology. 6 (6): 622–628. Bibcode:2003COPB....6..622E. doi:10.1016/j.pbi.2003.09.014. PMID   14611963.
  19. ↑ Chalfie M (October 1995). "Green fluorescent protein". Photochemistry and Photobiology. 62 (4): 651–656. doi:10.1111/j.1751-1097.1995.tb08712.x. PMID   7480149. S2CID   3944607.
  20. ↑ Grimm JB, English BP, Chen J, Slaughter JP, Zhang Z, Revyakin A, Patel R, Macklin JJ, Normanno D, Singer RH, Lionnet T, Lavis LD (March 2015). "A general method to improve fluorophores for live-cell and single-molecule microscopy". Nature Methods. 12 (3): 244–250. doi:10.1038/nmeth.3256. ISSN   1548-7091. PMC   4344395 . PMID   25599551.
  21. ↑ Huang B, Bates M, Zhuang X (2009-06-02). "Super-resolution fluorescence microscopy". Annual Review of Biochemistry. 78: 993–1016. doi:10.1146/annurev.biochem.77.061906.092014. PMC   2835776 . PMID   19489737.
  22. ↑ Leung BO, Chou KC (2011-09-01). "Review of Super-Resolution Fluorescence Microscopy for Biology". Applied Spectroscopy. 65 (9): 967–980. Bibcode:2011ApSpe..65..967L. doi:10.1366/11-06398. PMID   21929850. S2CID   5545465.
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