Immunohistochemistry

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Immunohistochemistry

• Department of pathology

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Introduction

• Immunohistochemistry (IHC) combines histological,
  immunological and biochemical techniques for the
  identification of specific tissue components by
  means of a specific antigen/antibody reaction
  tagged with a visible label. IHC makes it
  possible to visualize the distribution and
  localization of specific cellular components
  within a cell or tissue.

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History

• 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 1980s

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Principle

• The principle of immunohistochemistry is the
  localization of antigens in tissue sections by
  the use of labeled antibodies as specific
  reagents through antigen-antibody interactions
  that are visualized by a marker such as
  fluorescent dye, enzyme, radioactive element or
  colloidal gold.

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Method

• Direct Method
• Indirect Method
• PAP /APAAP Method
• ABC Method
• SP Method

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Direct Method
Labeled Antibody
Tissue Antigen
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Two-Step Indirect Method
Secondary Antibody
Primary Antibody
Tissue Antigen
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PAP Method (peroxidase anti-peroxidase method)
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ABC Method (avidin-biotin complex method )
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SP Method(streptavidin peroxidase conjugated
method)
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Applications

• Cancer diagnostics
• differential diagnosis
• Treatment of cancer
• Research

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General Immunohistochemistry Protocol
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Part 1
Tissue preparation

• 1.Fixation
• formalin fixation and paraffin embedding
• 2.Sectioning
• 3. Whole Mount Preparation

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Part 2
pretreatment

• 1. Antigen retrieval
• Proteolytic enzyme method and Heat-induced
  method
• 2. Inhibition of endogenous tissue components
• 3 H2O2, 0.01 avidin
• 3. Blocking of nonspecific sites
• 10 normal serum

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Part 3
staining

• Make a selection based on the type of specimen,
  the primary antibody, the degree
• of sensitivity and the processing time
  required as well as the cost of the reagents.

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SP Method(streptavidin peroxidase conjugated
method)
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Controls

• Positive Control
• It is to test for a protocol or procedure
  used.
• It will be ideal to use the tissue of known
  positive as a control.
• Negative Control
• It is to test for the specificity of the
  antibody involved.

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Example

• 1. Prepare and fix tissue
• 2. Antigen retrieval
• 3.Suppress endogenous peroxidase activity
• 4. Block nonspecific sites in the tissues
• 5. Incubate the tissues with the primary antibody
  
• 6. Incubate the tissues with the secondary
  antibody
• 7. Incubate the tissues with SP
• 8.Add DAB and incubate until desired staining is
  achieved

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Troubleshooting

• 1. Weak or No Staining
• 2. Over-staining
• 3. High Background

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Weak or No Staining
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Weak or No Staining
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Over-staining
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High Background
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Thank you !
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• Immunohistochemistry techniques have been used
  since the 1940s, when AH Coons and colleagues
  published their landmark paper describing the
  identification of tissue antigens using a direct
  fluorescence protocol (Coons AH, Creech HJ, Jones
  RN (1941) Immunological properties of an antibody
  containing a fluorescent group. Proc Soc Exp Biol
  Med 47200202).