Immunohistochemistry Techniques Overview

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Questions and Answers

What is the primary purpose of immunohistochemistry (IHC)?

  • To fix and preserve tissue samples
  • To visualize cellular structures using microscopy
  • To identify specific tissue components using antigen/antibody reactions (correct)
  • To process and section tissues for microscopic examination

Which of the following methods is a type of indirect immunohistochemistry?

  • PAP/APAAP Method (correct)
  • Direct Method
  • Heat-induced method
  • Formalin fixation

What is a common marker used in immunohistochemistry visualization?

  • Polymerase chain reaction
  • Gel electrophoresis
  • Colloidal gold (correct)
  • Methylene blue

What is the first step in the immunohistochemistry protocol?

<p>Prepare and fix tissue (A)</p> Signup and view all the answers

What role does the secondary antibody play in immunohistochemistry?

<p>It enhances the binding of the primary antibody (D)</p> Signup and view all the answers

Which of the following techniques is used for antigen retrieval?

<p>Heat-induced method (A)</p> Signup and view all the answers

Which application is NOT typically associated with immunohistochemistry?

<p>Genomics research (C)</p> Signup and view all the answers

What is the significance of performing both positive and negative controls in IHC?

<p>To confirm the specificity and reliability of the antibody reactions (A)</p> Signup and view all the answers

Which characteristic is NOT typically considered when selecting a primary antibody for IHC?

<p>Thickness of the tissue slice (A)</p> Signup and view all the answers

What is a primary difference between the direct and indirect methods of IHC?

<p>Indirect method requires multiple steps and secondary antibodies (B)</p> Signup and view all the answers

What is the primary function of the secondary antibody in the detection system?

<p>To bind to the primary antibody (C)</p> Signup and view all the answers

Which chromogen is most commonly used in applications for visualizing results under a microscope?

<p>3,3'-Diaminobenzidine (DAB) (A)</p> Signup and view all the answers

What can cause high background staining in tissue sections?

<p>Inadequate washing of sections (B)</p> Signup and view all the answers

Which type of antibody consists of a mixture of various antibody molecules and recognizes multiple epitopes?

<p>Polyclonal antibody (C)</p> Signup and view all the answers

What is a characteristic of DAB staining?

<p>It produces a brown color precipitate (C)</p> Signup and view all the answers

What factor can lead to weak or no staining in tissue samples?

<p>Inadequate antibody incubation time (B)</p> Signup and view all the answers

What type of counterstain is typically used alongside chromogenic detection?

<p>Hematoxylin (C)</p> Signup and view all the answers

What should be avoided to prevent non-specific binding of antibodies?

<p>High concentration of antibodies (B)</p> Signup and view all the answers

Which statement about primary antibodies is true?

<p>They can be polyclonal or monoclonal. (C)</p> Signup and view all the answers

What is a consequence of tissue over-fixation during processing?

<p>Diffusion of tissue antigen (B)</p> Signup and view all the answers

Flashcards

Immunohistochemistry (IHC)

A technique that uses antibodies to find specific parts in tissues.

IHC Principle

Using labeled antibodies to find antigens in tissue sections.

Direct IHC Method

Primary antibody is labeled and directly binds to the target.

Indirect IHC Method

Primary antibody binds; secondary antibody (labeled) recognizes it.

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IHC Application (Cancer)

Diagnosing cancer, comparing types, and research.

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Antigen Retrieval

Recovering tissue antigens (proteins) to make them findable.

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Positive Control

Samples with known targets used to verify IHC works.

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Negative Control

Samples without target to ensure false-positive detection isn't present.

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Tissue Fixation

Preserving tissue structure through chemical fixation for study.

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Secondary Antibody

Antibody that recognizes the first antibody.

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Target Antigen

Proteins found within or on the surface of a cell.

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Primary Antibody

Antibodies that bind to the target antigen.

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Chromogenic detection

Detection method using enzymes (like HRP) to produce a colored precipitate.

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3,3'-Diaminobenzidine (DAB)

A chromogen producing a brown colored precipitate.

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Alkaline Phosphatase (AP)

A chromogen producing a red colored precipitate.

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Inadequate deparaffinization

Insufficient removal of paraffin from tissue samples.

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Endogenous enzyme

Enzyme naturally present in the tissue.

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Non-specific binding

Antibodies binding to unintended sites in the tissue.

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High Background

Excessive staining outside the target area.

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Study Notes

Immunohistochemistry (IHC)

  • IHC is a combined histological, immunological, and biochemical technique
  • Used to identify specific tissue components
  • Achieved by a specific antigen-antibody reaction tagged with a visible label
  • Visualizes and localizes specific cellular components in cells or tissues

IHC Principle

  • Localizes antigens in tissue sections
  • Uses labeled antibodies targeting antigen-antibody interactions
  • Visual markers include fluorescent dyes, enzymes, radioactive elements, or colloidal gold

IHC Methods

  • Direct Method:
    • Uses a labeled primary antibody
    • Binds directly to the target antigen in tissue
  • Indirect Method:
    • Uses a labeled secondary antibody
    • Binds to the primary antibody, which is bound to the antigen
  • PAP/APAAP Method:
    • Uses peroxidase-antiperoxidase complex
  • ABC Method:
    • Uses avidin-biotin complex
  • SP Method:
    • Uses streptavidin peroxidase conjugate

Selection Considerations

  • Type of specimen
  • Primary antibody used
  • Sensitivity required
  • Processing time
  • Reagent cost

IHC Applications

  • Cancer diagnostics
  • Differential diagnosis
  • Cancer treatment
  • Research

IHC Protocol Overview

  • Part 1: Tissue Preparation
    • Fixation (formalin, paraffin embedding)
    • Tissue processing
    • Tissue sectioning
    • Whole-mount preparation
  • Part 2: Pretreatment
    • Antigen retrieval (heat-induced or proteolytic enzyme)
    • Inhibition of endogenous tissue components (3% Hâ‚‚Oâ‚‚ or 0.01% avidin)
    • Blocking of nonspecific sites (10% normal serum)
  • Part 3: Staining
    • Target antigens (proteins on or within cells)
    • Primary antibody (polyclonal or monoclonal)
    • Secondary antibody
    • Detection system (enzymes like horseradish peroxidase, HRP)
    • Chromogen (insoluble colored precipitate for visualization - DAB or AP)
    • Counter-stain (hematoxylin)

Controls

  • Positive Control:
    • Tissue with known positive result
    • Validates protocol and procedure
  • Negative Control:
    • Tests antibody specificity

Troubleshooting

  • Weak or No Staining:
    • Inadequate deparaffinization
    • Inactive primary antibodies
    • Low antibody concentration
    • Inadequate antibody incubation time
    • Improper or inadequate tissue fixation
    • Incompatible antibodies
    • Inactive secondary antibodies
    • Inadequate substrate incubation time
    • Incorrect reagent order/steps omitted
  • Over-staining:
    • Excessive antibody concentration
    • Excessively long incubation time
    • Too high incubation temperature
    • Long substrate incubation time
    • Dried-out sections
  • High Background:
    • Inadequate washing
    • Endogenous enzyme/biotin activity
    • Non-specific antibody binding
    • Diffusion of tissue antigen due to inadequate fixation

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Immunohistochemistry Lecture Notes PDF

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