IMMUNOHISTOCHEMISTRY (IHC)JESS AIMEE WHITE

What Is Immunohistochemistry?

• IHC Is a technique used in Neuroscience, which refers to the

detection of a specific antigen in cells using monoclonal and

polyclonal antibodies.

• Visualises the distribution and localisation of a specific

antigen or cellular component in separated tissues, or

tissue sections.

• Detects protein expression through the binding of an

antibody to a specific protein of interest

• IHC is an immunostaining technique

• Highly used in the diagnosis of abnormal cells, such as those

found in cancerous tumours.

• Can be used to identify cellular events – e.g. apoptosis

• Allows for the mapping of neurotransmitter distribution within

the nervous system – identifying neuronal circuits.

• High-affinity and highly specific binding by antibody probes

can map the spatial distribution of receptors, peptides, and

enzymes to a micrometre resolution.

What Is Immunohistochemistry?

• Visualising an antibody-antigen interaction can be

accomplished in a number of ways:

• An antibody can be conjugated to an enzyme, such as

peroxidase, that can catalyse a colour-producing reaction

• OR

• The antibody can also be tagged to a fluorophore, such as

fluorescein or rhodamine

What Is Immunohistochemistry?

• “Immunohistochemistry is an umbrella term that

encompasses many methods used to determine tissue

constituents (the antigens) with the employment of specific

antibodies that can be visualized through staining. When

used in cell preparations it is called immunocytochemistry, a

term that some authors use for all methods entailing the

immunological search of cell antigens, even when this

involves tissue slices.” (Matos et al., 2010)

Journal Definition

• The principle has existed since the 1930s.

• Started in 1941 when coons identified pneumococci using a direct

fluorescent method.

• Indirect method

• Addition of horseradish peroxidase

• Peroxidase anti-peroxidase technique in 1979

• Use of avidin & biotin complex in early 1980’s

History

• IHC is used for disease diagnosis, drug development and

biological research

• For Example:

• In tumour diagnosis (as benign or malignant)

• Also used in neuropathology and haematopathology

• Testing drug efficacy – by detecting either the activity or the up /

down regulation of disease targets.

Current Applications

• Predicts the prognosis of tumours by identification of enzymes,

tumour-specific antigens, oncogenes, tumour suppressor genes,

and tumour cell proliferation markers.

• Allows for clinical staging and histologic grading.

• Identifies the cell type and origin of a metastasis to find the site of

the primary tumour

Applications – Prognostic Markers in Cancer

• IHC methods have brought about a revolution in approach to

diagnosis of tumours of uncertain origin

• A variety of antibodies is chosen to resolve such diagnostic

problem cases.

• The selection of antibodies being made is based on clinical history

and morphological features

• Immunohistochemical stains for intermediate filaments are

expressed by tumour cells (keratin, desmin, vimentin,

neurofilaments, and glial fibrillary acidic proteins).

Applications – Tumours of Uncertain Histogenesis

• Immunohistochemical methods are also being applied to confirm

infectious agent in tissues by use of specific antibodies against

microbial DNA or RNA.

• For example, in cytomegalovirus, hepatitis B virus or hepatitis C

virus.

• Widely used for the detection of specific pathogens, viral as well as

bacterial and protozoal.

Applications – Infections

• Degenerative disorders of the nervous system include a wide range of diseases

characterized by the dysfunction and death of specific, selectively vulnerable

populations of nerve cells.

• Immunohistochemical staining for beta amyloid precursor protein has been

validated as a method to detect axonal injury.

• Neuroscience antibodies for IHC include:

http://www.enzolifesciences.com/fileadmin/files/minicatalog/Flyer_Neuroscien

ce-IHC-Antibodies.pdf

Applications – Neurodegenerative Disorders and Brain Trauma

• This technique is comprised of 2 phases:

• 1) Slide preparation (specimen fixation and tissue processing)

and the reaction (Antigen retrieval, non-specific site block,

endogenous peroxidase block, primary antibody incubation,

and the employment of systems of detection, revealing and

counterstaining and also slide mounting and storage)

• 2) Interpretation and quantification of the obtained expression.

• IHC requires the availability of biopsies

• These are processed into sections with a microtome and then the

sections are incubated with an appropriate antibody.

• The site of antibody binding is visualized under an ordinary or

fluorescent microscope by a marker such as a fluorescent dye,

enzymes or a radioactive element.

• This is directly linked to the primary antibody or to an appropriate

secondary antibody.

Requirements and Considerations

• Antibody selection

• Fixation

• Sectioning

• Antigen retrieval

• Blocking

• Controls

• Direct method

• Indirect method

• Immunoenzyme

• Fluorescence

• Multiple labeling

Requirements and Considerations

• Bias of the results.

• The acquisition, handling, fixation, specimen delivery to the

laboratory and antigen retrieval are all critical factors. Fresh

specimens that are inadvertently submitted to long periods of

fixation may significant lose antigenicity.

• For example, Jacobs et al., (1996) showed that there is progressive

loss of antigenicity upon only 12 week storage of breast cancer

histological slices on slides stored in ambient temperature for the

detection of p53, Bcl-2, oestrogen receptor and factor VIII proteins

Limitations, Difficulties & Problems

Neuroligin-2

Nestin

Serotonin

• Baek, J.H., Darlington, C.L., Smith, P.F. AND ASHTON, J.C. (2013) Antibody

Testing For Brain Immunohistochemistry: Brain Immunolabelling For The

Cannabinoid CB2 Receptor. Journal Of Neuroscience Methods. 216(2): 87-95

• Jacobs TW, Prioleau JE, Stillman IE, Et Al. Loss Of Tumour Marker-

immunostaining Intensity On Stored Paraffin Slides Of Breast Cancer. J Natl

Cancer Inst. 1996;88:1054–9.

• Matos, L.L., Trufelli, D.C., Matos, M.G.L. And Pinhal, M.A.S. (2010)

Immunohistochemistry As An Important Tool In Biomarkers Detection And

Clinical Practice. Biomarker Insights. 5: 9-20

• IHC Examples: Http://Www.Neuroscienceassociates.Com/Immuno.Htm

References