31:
226:
Flow cytometry has several advantages over IHC including: the ability to define distinct cell populations by their size and granularity; the capacity to gate out dead cells; improved sensitivity; and multi-colour analysis to measure several antigens simultaneously. However, flow cytometry can be less
294:
or cell/tissue extracts in a multi-well plate format (usually 96-wells per plate). Broadly, proteins in solution are absorbed to ELISA plates. Antibodies specific for the protein of interest are used to probe the plate. Background is minimised by optimising blocking and washing methods (as for IHC),
195:
methods that act by breaking some of the protein cross-links formed by fixation to uncover hidden antigenic sites. This can be accomplished by heating for varying lengths of times (heat induced epitope retrieval or HIER) or using enzyme digestion (proteolytic induced epitope retrieval or PIER).
199:
One of the main difficulties with IHC staining is overcoming specific or non-specific background. Optimisation of fixation methods and times, pre-treatment with blocking agents, incubating antibodies with high salt, and optimising post-antibody wash buffers and wash times are all important for
187:
sections do not require the tissue to be processed through organic solvents or high heat, which can destroy the antigenicity, or disrupted by freeze thawing. The disadvantage of vibratome sections is that the sectioning process is slow and difficult with soft and poorly fixed tissues, and that
312:
or EM can be used to study the detailed microarchitecture of tissues or cells. Immuno-EM allows the detection of specific proteins in ultrathin tissue sections. Antibodies labelled with heavy metal particles (e.g. gold) can be directly visualised using
457:
In laboratory science, immunostaining can be used for a variety of applications based on investigating the presence or absence of a protein, its tissue distribution, its sub-cellular localisation, and of changes in protein expression or degradation.
227:
effective at detecting extremely rare cell populations, and there is a loss of architectural relationships in the absence of a tissue section. Flow cytometry also has a high capital cost associated with the purchase of a flow cytometer.
169:
is essential for the preservation of cell morphology and tissue architecture. Inappropriate or prolonged fixation may significantly diminish the antibody binding capability. Many antigens can be successfully demonstrated in
182:
and cut with a cryostat. The disadvantages of frozen sections include poor morphology, poor resolution at higher magnifications, difficulty in cutting over paraffin sections, and the need for frozen storage. Alternatively,
477:
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can be used for the direct analysis of cells expressing one or more specific proteins. Cells are immunostained in solution using methods similar to those used for immunofluorescence, and then analysed by flow cytometry.
317:. While powerful in detecting the sub-cellular localisation of a protein, immuno-EM can be technically challenging, expensive, and require rigorous optimisation of tissue fixation and processing methods. Protein
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via dry, semi-dry, or wet blotting methods. The membrane can then be probed using antibodies using methods similar to immunohistochemistry, but without a need for fixation. Detection is typically performed using
268:
Western blotting is a routine molecular biology method that can be used to semi-quantitatively compare protein levels between extracts. The size separation prior to blotting allows the protein
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178:-embedded tissue sections. However, some antigens will not survive even moderate amounts of aldehyde fixation. Under these conditions, tissues should be rapidly fresh frozen in
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586:
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The primary antibody can be labeled using a small molecule which interacts with a high affinity binding partner that can be linked to an enzyme or fluorophore. The
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was proposed to alleviate the problems caused by frequent incompatibility of antibody staining with fixation protocols that better preserve cell morphology.
877:
286:
The enzyme-linked immunosorbent assay or ELISA is a diagnostic method for quantitatively or semi-quantitatively determining protein concentrations from
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and specificity is ensured via the presence of positive and negative controls. Detection methods are usually colorimetric or chemiluminescence based.
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are now used. These enzymes are capable of catalysing reactions that give a coloured product that is easily detectable by light
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Western blotting allows the detection of specific proteins from extracts made from cells or tissues, before or after any
852:
66:
951:
857:
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496:
Coons, Albert; Creech HJ; Jones, RN (1941). "Immunological properties of an antibody containing a fluorescent group".
30:
304:
809:
799:
789:
751:
652:"Use of Protein Biotinylation in Vivo for Immunoelectron Microscopic Localization of a Specific Protein Isoform"
995:
829:
702:
398:, antibodies are linked to a heavy metal particle (typically gold nanoparticles in the range 5-15nm diameter).
116:), is perhaps the most commonly applied immunostaining technique. While the first cases of IHC staining used
990:
472:
615:
Cherie, H (2004). "Applications of Flow
Cytometry and Immunohistochemistry to Diagnostic Hematopathology".
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obtaining high quality immunostaining. In addition, the presence of both positive and negative
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in 1941. However, immunostaining now encompasses a broad range of techniques used in
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Viens, A.; Harper, F.; Pichard, E.; Comisso, M.; Pierron, G.; Ogryzko, V. (2008).
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The applications of immunostaining are numerous, but are most typically used in
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for the diagnosis of specific types of cancers based on molecular markers.
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in a sample. The term "immunostaining" was originally used to refer to the
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The primary antibody can be probed for using a broader species-specific
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List of histologic stains that aid in diagnosis of cutaneous conditions
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chatter marks or vibratome lines are often apparent in the sections.
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Ramos-Vara, JA (2005). "Technical
Aspects of Immunohistochemistry".
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can be used as labels, and the immunoreaction can be visualized by
887:
281:
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are commonly used to catalyse reactions that give a coloured or
698:
191:
The detection of many antigens can be dramatically improved by
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to be gauged as compared with known molecular weight markers.
120:
587:"IHC Tip 1: Antigen retrieval - should I do PIER or HIER?"
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for staining are essential for determining specificity.
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The primary antibody can be directly labeled using an
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steps. Proteins are generally separated by size using
468:
Cutaneous conditions with immunofluorescence findings
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391:that is labeled using an enzyme, or fluorophore.
382:is one commonly used high affinity interaction.
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617:Archives of Pathology and Laboratory Medicine
8:
656:Journal of Histochemistry and Cytochemistry
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703:
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85:that use antibody-based staining methods.
69:of tissue sections, as first described by
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591:Bio-Rad Antibodies (formerly AbD Serotec)
402:As previously described, enzymes such as
104:Immunohistochemistry or IHC staining of
29:
488:
129:), other non-fluorescent methods using
960:Photoactivated localization microscopy
878:Protein–protein interaction prediction
359:can be accomplished in multiple ways.
7:
835:Freeze-fracture electron microscopy
61:-based method to detect a specific
421:molecules can be visualised using
25:
574:Immunohistochemistry Introduction
276:Enzyme-linked immunosorbent assay
815:Isothermal titration calorimetry
795:Dual-polarization interferometry
315:transmission electron microscopy
261:linked antibodies to catalyse a
332:In immunostaining methods, an
249:before being transferred to a
1:
805:Chromatin immunoprecipitation
355:. Detection of this first or
336:is used to detect a specific
868:Protein structural alignment
853:Protein structure prediction
629:10.5858/2004-128-1004-AOFCAI
67:immunohistochemical staining
952:Super-resolution microscopy
858:Protein function prediction
786:Peptide mass fingerprinting
781:Protein immunoprecipitation
450:Clinically, IHC is used in
112:, which is the staining of
41:immunostained section of a
1012:
510:10.3181/00379727-47-13084P
343:. These antibodies can be
305:immune electron microscopy
302:
299:Immuno-electron microscopy
279:
234:
211:
97:
810:Surface plasmon resonance
800:Microscale thermophoresis
790:Protein mass spectrometry
752:Green fluorescent protein
140:immunoperoxidase staining
830:Cryo-electron microscopy
863:Protein–protein docking
776:Protein electrophoresis
668:10.1369/jhc.2008.951624
473:Immunostaining protocol
423:fluorescence microscopy
328:Methodological overview
762:Protein immunostaining
585:AbD Serotec, Bio-Rad.
165:Tissue preparation or
46:
820:X-ray crystallography
498:Proc Soc Exp Biol Med
33:
27:Biochemical technique
747:Protein purification
441:clinical diagnostics
411:alkaline phosphatase
145:alkaline phosphatase
100:Immunohistochemistry
94:Immunohistochemistry
772:Gel electrophoresis
545:10.1354/vp.42-4-405
445:laboratory research
428:confocal microscopy
396:electron microscopy
310:Electron microscopy
247:gel electrophoresis
110:immunocytochemistry
915:Display techniques
767:Protein sequencing
388:secondary antibody
151:. Alternatively,
126:immunofluorescence
47:
973:
972:
922:Bacterial display
193:antigen retrieval
83:molecular biology
57:is any use of an
16:(Redirected from
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937:Ribosome display
873:Protein ontology
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623:(9): 1004–1022.
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593:. Archived from
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415:chemiluminescent
357:primary antibody
270:molecular weight
263:chemiluminescent
231:Western blotting
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996:Protein methods
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906:Secretion assay
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662:(10): 911–919.
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394:In the case of
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180:liquid nitrogen
160:autoradiography
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539:(4): 405–426.
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452:histopathology
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303:Main article:
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280:Main article:
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235:Main article:
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220:flow cytometer
214:Flow cytometry
212:Main article:
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208:Flow cytometry
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98:Main article:
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55:immunostaining
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942:Yeast display
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932:Phage display
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901:Protein assay
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597:on 2016-04-23
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927:mRNA display
896:Enzyme assay
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757:Western blot
739:Experimental
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599:. Retrieved
595:the original
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435:Applications
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380:streptavidin
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288:blood plasma
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243:purification
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237:Western blot
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124:
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79:cell biology
71:Albert Coons
54:
51:biochemistry
48:
43:brain tumour
965:Vertico SMI
825:Protein NMR
419:Fluorescent
404:horseradish
369:fluorophore
153:radioactive
118:fluorescent
18:Immunostain
986:Immunology
980:Categories
601:2017-01-05
533:Vet Pathol
484:References
407:peroxidase
352:polyclonal
346:monoclonal
265:reaction.
259:peroxidase
149:microscopy
135:peroxidase
89:Techniques
35:Micrograph
518:101356912
417:product.
251:synthetic
185:vibratome
75:histology
732:of study
726:Proteins
686:18574249
637:15335254
553:16006601
462:See also
334:antibody
254:membrane
202:controls
176:paraffin
172:formalin
167:fixation
156:elements
133:such as
59:antibody
730:methods
677:2544619
561:6229029
341:epitope
338:protein
322:in vivo
174:-fixed
131:enzymes
63:protein
728:: key
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635:
559:
551:
516:
376:biotin
365:enzyme
143:) and
106:tissue
81:, and
888:Assay
557:S2CID
514:S2CID
292:serum
282:ELISA
137:(see
123:(see
114:cells
37:of a
682:PMID
633:PMID
549:PMID
443:and
121:dyes
39:GFAP
672:PMC
664:doi
625:doi
621:128
541:doi
506:doi
425:or
409:or
367:or
349:or
49:In
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Text is available under the Creative Commons Attribution-ShareAlike License. Additional terms may apply.
