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?

Prepare and fix tissue Signup and view all the answers

What role does the secondary antibody play in immunohistochemistry?

It enhances the binding of the primary antibody Signup and view all the answers

Which of the following techniques is used for antigen retrieval?

Heat-induced method Signup and view all the answers

Which application is NOT typically associated with immunohistochemistry?

Genomics research Signup and view all the answers

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

To confirm the specificity and reliability of the antibody reactions Signup and view all the answers

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

Thickness of the tissue slice Signup and view all the answers

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

Indirect method requires multiple steps and secondary antibodies Signup and view all the answers

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

To bind to the primary antibody Signup and view all the answers

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

3,3'-Diaminobenzidine (DAB) Signup and view all the answers

What can cause high background staining in tissue sections?

Inadequate washing of sections Signup and view all the answers

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

Polyclonal antibody Signup and view all the answers

What is a characteristic of DAB staining?

It produces a brown color precipitate Signup and view all the answers

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

Inadequate antibody incubation time Signup and view all the answers

What type of counterstain is typically used alongside chromogenic detection?

Hematoxylin Signup and view all the answers

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

High concentration of antibodies Signup and view all the answers

Which statement about primary antibodies is true?

They can be polyclonal or monoclonal. Signup and view all the answers

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

Diffusion of tissue antigen Signup and view all the answers

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% H₂O₂ 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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Description

Explore the principles, methods, and applications of immunohistochemistry (IHC) in this quiz. Learn about the various techniques such as direct and indirect methods, as well as specialized approaches like the PAP and ABC methods. This quiz is designed for those interested in histological and immunological analysis.