20:
215:
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
283:
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),
184:
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).
188:
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
176:
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
301:
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
446:
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.
216:
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.
158:
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
171:
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,
211:
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.
306:. 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
245:
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
466:
257:
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
167:-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
575:
363:
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
456:
313:
was proposed to alleviate the problems caused by frequent incompatibility of antibody staining with fixation protocols that better preserve cell morphology.
813:
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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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705:
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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
948:
230:
Western blotting allows the detection of specific proteins from extracts made from cells or tissues, before or after any
485:
Coons, Albert; Creech HJ; Jones, RN (1941). "Immunological properties of an antibody containing a fluorescent group".
803:
783:
303:
764:
27:
641:"Use of Protein Biotinylation in Vivo for Immunoelectron Microscopic Localization of a Specific Protein Isoform"
387:, antibodies are linked to a heavy metal particle (typically gold nanoparticles in the range 5-15nm diameter).
793:
698:
105:), is perhaps the most commonly applied immunostaining technique. While the first cases of IHC staining used
823:
841:
604:
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
39:
31:
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The primary antibody can be probed for using a broader species-specific
727:
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List of histologic stains that aid in diagnosis of cutaneous conditions
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123:
23:
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chatter marks or vibratome lines are often apparent in the sections.
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63:
520:
Ramos-Vara, JA (2005). "Technical
Aspects of Immunohistochemistry".
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can be used as labels, and the immunoreaction can be visualized by
47:
714:
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51:
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are commonly used to catalyse reactions that give a coloured or
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353:
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The detection of many antigens can be dramatically improved by
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to be gauged as compared with known molecular weight markers.
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270:
576:"IHC Tip 1: Antigen retrieval - should I do PIER or HIER?"
638:
109:
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for staining are essential for determining specificity.
484:
352:
The primary antibody can be directly labeled using an
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steps. Proteins are generally separated by size using
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Cutaneous conditions with immunofluorescence findings
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713:
966:
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380:that is labeled using an enzyme, or fluorophore.
371:is one commonly used high affinity interaction.
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606:Archives of Pathology and Laboratory Medicine
287:
513:
645:Journal of Histochemistry and Cytochemistry
478:
706:
692:
519:
316:
74:that use antibody-based staining methods.
58:of tissue sections, as first described by
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580:Bio-Rad Antibodies (formerly AbD Serotec)
391:As previously described, enzymes such as
93:Immunohistochemistry or IHC staining of
18:
597:
573:
118:), other non-fluorescent methods using
82:
967:
949:Photoactivated localization microscopy
867:Protein–protein interaction prediction
603:
348:can be accomplished in multiple ways.
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824:Freeze-fracture electron microscopy
219:
50:-based method to detect a specific
13:
410:molecules can be visualised using
14:
996:
563:Immunohistochemistry Introduction
265:Enzyme-linked immunosorbent assay
196:
804:Isothermal titration calorimetry
784:Dual-polarization interferometry
304:transmission electron microscopy
250:linked antibodies to catalyse a
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632:
567:
321:In immunostaining methods, an
238:before being transferred to a
1:
794:Chromatin immunoprecipitation
472:
344:. Detection of this first or
325:is used to detect a specific
77:
857:Protein structural alignment
842:Protein structure prediction
618:10.5858/2004-128-1004-AOFCAI
56:immunohistochemical staining
7:
941:Super-resolution microscopy
847:Protein function prediction
775:Peptide mass fingerprinting
770:Protein immunoprecipitation
450:
439:Clinically, IHC is used in
101:, which is the staining of
30:immunostained section of a
10:
1001:
499:10.3181/00379727-47-13084P
332:. These antibodies can be
294:immune electron microscopy
291:
288:Immuno-electron microscopy
268:
223:
200:
86:
939:
903:
875:
832:
799:Surface plasmon resonance
789:Microscale thermophoresis
779:Protein mass spectrometry
741:Green fluorescent protein
726:
129:immunoperoxidase staining
819:Cryo-electron microscopy
852:Protein–protein docking
765:Protein electrophoresis
657:10.1369/jhc.2008.951624
462:Immunostaining protocol
412:fluorescence microscopy
317:Methodological overview
751:Protein immunostaining
574:AbD Serotec, Bio-Rad.
154:Tissue preparation or
35:
809:X-ray crystallography
487:Proc Soc Exp Biol Med
22:
16:Biochemical technique
736:Protein purification
430:clinical diagnostics
400:alkaline phosphatase
134:alkaline phosphatase
89:Immunohistochemistry
83:Immunohistochemistry
761:Gel electrophoresis
534:10.1354/vp.42-4-405
434:laboratory research
417:confocal microscopy
385:electron microscopy
299:Electron microscopy
236:gel electrophoresis
99:immunocytochemistry
904:Display techniques
756:Protein sequencing
377:secondary antibody
140:. Alternatively,
115:immunofluorescence
36:
962:
961:
911:Bacterial display
182:antigen retrieval
72:molecular biology
46:is any use of an
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926:Ribosome display
862:Protein ontology
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612:(9): 1004–1022.
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582:. Archived from
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404:chemiluminescent
346:primary antibody
259:molecular weight
252:chemiluminescent
220:Western blotting
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985:Protein methods
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895:Secretion assay
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651:(10): 911–919.
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383:In the case of
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169:liquid nitrogen
149:autoradiography
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980:Flow cytometry
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834:Bioinformatics
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528:(4): 405–426.
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493:(2): 200–202.
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441:histopathology
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292:Main article:
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269:Main article:
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224:Main article:
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209:flow cytometer
203:Flow cytometry
201:Main article:
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197:Flow cytometry
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87:Main article:
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44:immunostaining
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931:Yeast display
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921:Phage display
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890:Protein assay
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586:on 2016-04-23
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916:mRNA display
885:Enzyme assay
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746:Western blot
728:Experimental
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588:. Retrieved
584:the original
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424:Applications
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369:streptavidin
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277:blood plasma
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232:purification
229:
226:Western blot
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127:
113:
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68:cell biology
60:Albert Coons
43:
40:biochemistry
37:
32:brain tumour
954:Vertico SMI
814:Protein NMR
408:Fluorescent
393:horseradish
358:fluorophore
142:radioactive
107:fluorescent
975:Immunology
969:Categories
590:2017-01-05
522:Vet Pathol
473:References
396:peroxidase
341:polyclonal
335:monoclonal
254:reaction.
248:peroxidase
138:microscopy
124:peroxidase
78:Techniques
24:Micrograph
507:101356912
406:product.
240:synthetic
174:vibratome
64:histology
721:of study
715:Proteins
675:18574249
626:15335254
542:16006601
451:See also
323:antibody
243:membrane
191:controls
165:paraffin
161:formalin
156:fixation
145:elements
122:such as
48:antibody
719:methods
666:2544619
550:6229029
330:epitope
327:protein
311:in vivo
163:-fixed
120:enzymes
52:protein
717:: key
673:
663:
624:
548:
540:
505:
365:biotin
354:enzyme
132:) and
95:tissue
70:, and
877:Assay
546:S2CID
503:S2CID
281:serum
271:ELISA
126:(see
112:(see
103:cells
26:of a
671:PMID
622:PMID
538:PMID
432:and
110:dyes
28:GFAP
661:PMC
653:doi
614:doi
610:128
530:doi
495:doi
414:or
398:or
356:or
338:or
38:In
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34:.
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