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L.10 IHC Flashcards

1
Q

What are Immunocytochemistry and Immunohistochemistry (IHC)?

A

Techniques that use labeled antibodies to detect specific antigens within cells or tissue sections.

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2
Q

What does ‘in situ’ mean in the context of immunostaining?

A

Shows the exact location of the target antigen in the tissue or cell.

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3
Q

What is a key characteristic of antibodies used in immunohistochemistry?

A

Highly specific: Antibodies are designed to bind only to their unique antigen.

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4
Q

What advantage does immunohistochemistry have regarding antigen detection?

A

Sensitive: Small amounts of antigen can be detected.

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5
Q

List four applications of immunohistochemistry.

A
  • Tumor typing
  • Infection detection
  • Immunological studies
  • Research applications
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6
Q

How does the primary antibody function in immunohistochemistry?

A

Binds specifically to the antigen in the tissue.

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7
Q

What is typically involved in the detection system of immunohistochemistry?

A

A chromogen and a label (e.g., enzyme or fluorophore) to visualize the antigen-antibody complex.

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8
Q

What is the purpose of antigen retrieval in tissue pretreatment?

A

To unmask antigens and enhance antibody binding.

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9
Q

What are the two main methods of antigen retrieval?

A
  • Proteolytic Digestion
  • Heat-Induced Epitope Retrieval (HIER)
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10
Q

What enzymes are commonly used in proteolytic digestion?

A
  • Trypsin
  • Protease
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11
Q

What does Heat-Induced Epitope Retrieval (HIER) involve?

A

Heating tissue sections in a specific buffer.

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12
Q

What pH buffers are typically used in HIER?

A
  • pH 6 (citrate buffer)
  • pH 9 (EDTA buffer)
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13
Q

What methods can be used to perform HIER?

A
  • Microwave
  • Pressure cooker
  • Water bath
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14
Q

True or False: Different antigens may respond equally to all retrieval methods.

A

False: Different antigens may respond better to different retrieval methods.

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15
Q

What must be tested to ensure proper antibody validation?

A

Multiple retrieval conditions.

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16
Q

What is the purpose of hydrogen peroxide blocking in immunostaining?

A

Quenches endogenous peroxidase activity

This prevents false positive brown staining when using DAB as a chromogen.

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17
Q

Why is hydrogen peroxide blocking important?

A

Prevents unwanted staining from red blood cells and other blood elements

This reduces background noise in the slide.

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18
Q

What is the function of normal serum blocking?

A

Blocks non-specific protein binding sites and prevents non-specific binding of the secondary antibody

Serum contains albumin, which effectively binds to open protein sites.

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19
Q

What type of serum should be used for blocking?

A

Serum from the host species of the secondary antibody

For example, if your secondary antibody is from a horse, use horse serum.

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20
Q

What are the benefits of using automated immunostaining systems?

A

Rapid, consistent, and high-throughput staining protocols

Automation enhances sensitivity and reduces background.

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21
Q

List three major commercial automated immunostaining systems.

A
  • Roche Ventana
  • Dako (Agilent)
  • Leica Biosystems
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22
Q

What is a key advantage of modern automated platforms in immunostaining?

A

Standardized protocols, batch processing, and reproducibility

These are essential for clinical diagnostics.

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23
Q

Fill in the blank: Serum is applied before the primary antibody to block _______.

A

[non-specific protein binding sites]

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24
Q

True or False: Normal serum blocking prevents specific antigen-antibody interactions.

A

False

It ensures that only specific interactions are detected.

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25
What animals are commonly used to produce antibodies?
Mice and rabbits ## Footnote These animals are preferred due to their immunological characteristics.
26
What stimulates an immune response in animals for antibody production?
Injection of purified antigen ## Footnote The antigen is a substance that induces an immune response.
27
What are the two main types of antibodies that can be harvested?
Polyclonal Antibodies and Monoclonal Antibodies ## Footnote Each type has distinct production methods and characteristics.
28
What are polyclonal antibodies collected from?
Animal's serum ## Footnote They contain a mixture of antibodies.
29
What is a key advantage of polyclonal antibodies?
Strong signal due to multiple binding ## Footnote This results from their ability to recognize multiple epitopes.
30
What is a disadvantage of polyclonal antibodies?
Risk of cross-reactivity and non-specific staining ## Footnote This can lead to false positives in tests.
31
How are monoclonal antibodies produced?
Via Hybridoma Technology ## Footnote This involves fusing B-cells with myeloma cells.
32
What do hybridomas produce?
One specific antibody against a single epitope ## Footnote This results in high specificity.
33
What is a key advantage of monoclonal antibodies?
High specificity and consistency between batches ## Footnote They provide reliable results across experiments.
34
What is a disadvantage of monoclonal antibodies?
Narrower range of antigen recognition ## Footnote They may not bind to all variants of an antigen.
35
What is the function of a primary antibody in immunostaining?
Binds directly to the target antigen/epitope ## Footnote It can be monoclonal or polyclonal.
36
What does a secondary antibody do?
Binds to the primary antibody and carries the detection label ## Footnote It amplifies the signal for detection.
37
What should secondary antibodies be raised against?
The species of the primary antibody ## Footnote This ensures proper binding.
38
What is the most commonly used antibody class in immunostaining?
IgG ## Footnote IgG antibodies are known for their versatility.
39
What is a defining characteristic of monoclonal antibodies?
Bind a single epitope (high specificity) ## Footnote This makes them reliable for specific target detection.
40
What is a defining characteristic of polyclonal antibodies?
Bind multiple epitopes (higher signal, but higher risk of background) ## Footnote This can be advantageous in certain assays.
41
Why is validation of antibodies crucial before clinical testing?
To ensure accuracy and reliability of results ## Footnote This minimizes the risk of erroneous conclusions.
42
What is the purpose of detection methods in immunostaining?
Enhance the visibility of the antigen-antibody reaction by using labels like enzymes or fluorochromes.
43
What is the direct method in immunostaining?
The label (enzyme or fluorochrome) is directly attached to the primary antibody.
44
What is a pro of the direct method?
Quick (1 step).
45
What are two cons of the direct method?
* Expensive * Higher background noise
46
What is the typical use of the direct method?
Typically used for direct immunofluorescence.
47
What are the indirect methods in immunostaining?
Amplify the signal by layering: Primary antibody → Secondary antibody (labelled) → Detection system.
48
What is preferred for tissue staining in immunostaining?
Indirect (Two-Step or Multi-Step) Methods.
49
What are the two main strategies in indirect methods?
* Avidin-Biotin Complex (ABC) Methods * Polymer-Based Detection Methods
50
What does the Avidin-Biotin Complex (ABC) Method use?
Biotinylated secondary antibodies that bind to a reporter enzyme (e.g., peroxidase) via avidin.
51
What are the components of the ABC method?
* Biotinylated secondary antibody * Avidin (or streptavidin in modified systems) linked to an enzyme
52
What is the Labelled Streptavidin-Biotin (LSAB) method?
A variant of ABC method using streptavidin instead of avidin to reduce non-specific binding.
53
What is a challenge associated with biotin-based detection systems?
Endogenous biotin can cause false positive staining.
54
What is required to avoid artefacts in biotin-based detection systems?
Biotin blocking steps.
55
What are polymer-based non-biotin detection systems designed to overcome?
Issues with endogenous biotin ## Footnote Endogenous biotin can interfere with detection methods in immunohistochemistry.
56
What were early polymer systems based on?
Dextran backbones with multiple enzymes and secondary antibodies attached ## Footnote These early systems aimed to enhance detection capabilities.
57
What are some advantages of modern polymer systems?
* Greater sensitivity due to better tissue penetration * Reduced background from avoiding biotin * No need for blocking endogenous biotin
58
Name an example of a polymer-HRP conjugate detection system.
EnVision™ (Dako) ## Footnote EnVision™ utilizes polymer-based technology for enhanced detection.
59
What is the principle of visualization in IHC?
Labels detect and localize antigen-antibody binding in situ after antibody binding ## Footnote This process is essential for visualizing specific proteins in tissue samples.
60
List some types of labels that can be used to visualize antigen-antibody complexes.
* Radioactive isotopes * Enzymes * Biotin * Metals * Oligonucleotides
61
What do chromogenic detection methods rely on?
Enzymes attached to the antibody complex that catalyze a reaction to form a colored product ## Footnote This is a common method used in immunohistochemistry.
62
What is the product color when using Horseradish Peroxidase (HRP) with DAB?
Brown ## Footnote This color indicates the presence of the antigen at the site of binding.
63
What is the substrate used in the HRP reaction with DAB?
DAB (3,3'-diaminobenzidine) + H₂O₂ ## Footnote This combination is crucial for producing the colored product.
64
What is the role of hydrogen peroxide in the HRP reaction?
Acts as a substrate for the HRP enzyme ## Footnote It facilitates the breakdown to release oxygen, which then reacts with DAB.
65
What is formed at the site of antigen binding in the HRP and DAB reaction?
Insoluble brown polymer ## Footnote This is the visible indication of the antigen's presence.
66
List advantages of chromogenic products.
* Permanent * Sections can be archived for long-term storage * More sensitive than fluorescence-based detection
67
What is the role of Alkaline Phosphatase in immunostaining?
Catalyzes a chemical reaction with substrates like ACE, forming a red precipitate at the site of the antigen. ## Footnote Often used when a different colour is desired or when there is high endogenous peroxidase, such as in blood-rich tissues.
68
What is the purpose of counterstaining in immunohistochemistry?
Provides tissue context by staining cell structures, enabling better visual contrast between positive staining and background.
69
Name a common counterstain used in immunohistochemistry.
Mayer’s Haematoxylin. ## Footnote It stains nuclei (DNA) blue/violet after 'bluing'.
70
What is the bluing process in histology?
Rinsing slides in running tap water to increase pH, converting nuclei from purple to blue/violet.
71
What is the purpose of a positive control in immunohistochemistry?
Confirms the staining procedure worked by using tissue known to express the target antigen.
72
What is the purpose of a negative control in immunohistochemistry?
Checks for non-specific staining by applying no primary antibody to the tissue.
73
True or False: Negative controls help detect background staining caused by non-specific binding.
True.
74
What is an application of immunostaining for Oestrogen receptor (ER) in breast tumour cells?
Breast cancer prognosis and treatment planning.
75
Fill in the blank: Methyl Green is a counterstain that targets _______.
nucleic acids.
76
What method is used for staining tumour cells in clusters?
Immunoalkaline phosphatase (AP + Red chromogen).
77
What is the target in the immunostaining application for tumour cells in clusters?
Various tumour markers.

Cell Pathology (28 decks)

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