An overview of the biological technique immunohistochemistry, with a neuroscience basis.

1. IMMUNOHISTOCHEMISTRY (IHC)
JESS AIMEE WHITE

2. 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

3. • 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?

4. • 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?

5. • “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

6. • 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

7. • 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

8. • 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

9. • 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

10. • 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

11. • 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

12. • 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.

13. • 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

14. • Antibody selection
• Fixation
• Sectioning
• Antigen retrieval
• Blocking
• Controls
• Direct method
• Indirect method
• Immunoenzyme
• Fluorescence
• Multiple labeling
Requirements and Considerations

15. • 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

16. Neuroligin-2

17. Nestin

18. Serotonin

19. • 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