Methods Of Immunostaining

1. IMMUNOSTAINING
SOUVIK BISWAS
M.PHARM (PHARMACOLOGY), 1ST YEAR (2ND SEM)
ROLL NO. 27720216003
REGISTRATION NO.162772310018
UNDER THE GUIDANCE OF DR. SANDIPAN DASGUPTA
NSHM KNOWLEDGE CAMPUS, KOLKATA – GROUP OF INSTITUTION

2. ANTIGENS
Antigen is any substance (such as
an immunogen or a hapten) foreign
to the body that evokes an immune
response either alone or after
forming a complex with a larger
molecule (such as a protein) and
that is capable of binding with a
product (such as an antibody or T
cell) of the immune response.

3. ANTIBODIES (IMMUNOGLOBULINS; IG)
It is a large number of proteins of high
molecular weight that are produced
normally by specialized B cells after
stimulation by an antigen and act
specifically against the antigen in an
immune response and that typically
consist of four subunits including two
heavy chains and two light chains -
called also immunoglobulin.

4. WHY DO IMMUNOSTAINING?
• Confirm genotype-phenotype
(e.g. KO, KI, levels or efficiency of expression, etc.)
• Where is a biomarker expressed?
• When is a biomarker expressed?
• Co-expression or co-localization.
• Cell type-specific biomarkers
(e.g. CD4, CD8 T-cells, NK, Macrophages, Mast Cells, etc.)

5. INTRODUCTION AND DEFINITION
• Immunostaining is a general term in
biochemistry that applies to 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.
• The key to immunohistochemistry is the
specific antibody-mediated detection of a
target antigen, known as immunostaining or
immunodetection.

6. IMMUNOSTAINING METHOD
According to different biotins conjugated with antibodies, IHC staining
methods can be classified as
• Immunofluorescence,
• Immunoenzymological staining
• Immunocolloidal gold technique
According to different kinds of procedures, immunohistochemistry staining
can be divided into subtypes of
• Direct staining (one-step staining)
• Indirect staining (two-step, three-step or multi-step staining)
• PAP staining method

7. DIFFERENT BIOTINS CONJUGATED WITH
ANTIBODIES
• Immunofluorescence:
It is the first immunohistochemical staining
method. With antigen-antibody binding reaction,
antigens are visualized by fluorescence dyes
conjugated with antibodies and that is localized
under fluorescence microscope.
• Immunoenzymological Staining:
In here enzyme-labeled antibodies are used
to bind with specific antigens in tissues samples
or cultured cells and localized by light microscope.

8. CONTD…
• Immunocolloidal Gold Technique:
It is a kind of technique that uses
colloidal gold as a marker.
Immunocolloidal gold technique is
suitable for single or multi-label
detection under immune-electron
microscope, and light microscope.

9. DIFFERENT KINDS OF PROCEDURES
• Direct Staining:
Incubate the sections with mixture of two
primary antibodies which are respectively
conjugated with two fluorescence dyes (e.g. FITC
and TRITC) Or, successively incubate sections
with two primary antibodies.
• Indirect Staining:
In here the primary antibodies are without
fluorescence dyes. Incubate sections with one
kind of primary antibody and corresponding
secondary antibody, then the other.

10. CONTD…
• PAP Staining Method (peroxidase
anti-peroxidase method):
Incubate sections with
corresponding secondary antibodies
which are conjugated with two
different enzymes (e.g. HRP, AKP), or
anti-HRP (PAP complex), anti-AKP
(APAAP complex).

11. Immunostaining
Protocols

12. IMMUNOHISTOCHEMISTRY STAINING TIPS
• Fresh fixed tissue
• Sufficient dehydration of tissues
• An intact, uniform and smooth sectioning
• Binder materials
• Sufficient deparaffinization
• A thorough inhibition of endogenous peroxidase

13. APPLICATIONS
• Clinically, IHC is used in histopathology for the diagnosis of
specific types of cancers based on molecular markers.
• 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
localization, and of changes in protein expression or
degradation.

14. REFERENCES
• Formation of the first cleavage spindle in nematode embryos by Albertson,
D.GDev. Biol. 101, (1984). page no. 61–72.
• Immunohistochemistry: Methods Express, edited by Simon Renshaw, Scion
Publishing Ltd, Bloxham, UK, 1st edition (15 Dec. 2006), page no. 45-96
• http://www.abcam.com/index.html?pageconfig=resource&rid=12615
(accessed on 16.05.2017)
• http://www.mdbioproducts.com/resources/protocols/immunohistochemistr
y (accessed on 17.05.2017)
• http://www.immunohistochemistry.us/immunohistochemistry-staining.html
(accessed on 18.05.2017)