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2.2 Cellular Pathology > Immunohistochemistry > Flashcards

Immunohistochemistry Flashcards

1
Q

What is immunocyto/histochemistry

A

An indirect staining method using labelled antibodies to detect specific antigens/epitopes in cells/tissues

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2
Q

What are four characteristics of immunostaining

A

In situ -> shows the location of the component in cell/tissue

Specific

Sensitive

Valuable in tumour typing, immunology and research

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3
Q

How does immunostaining work?

A

The antibody attaches to the cell/tissue antigen, it is then visualised with a chromogen through a label which enables visualisation of the ag/ab complexx

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4
Q

What must be done prior to immunostaining

A

Antigen retrieval

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5
Q

What are the two main antigen retrieval methods?

A

Proteolytic digestion

Heat induced epitope retrieval (HIER)

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6
Q

What is meant by proteolytic digestion for antigen retrieval

A

This is where tissue sections are incubated with an enzyme e.g. trypsin or protease

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7
Q

What is meant by HIER antigen retrieval

A

HIER uses either a pressure cooker or a microwave

Slides are placed in a buffer of either pH6 or pH9 and heated

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8
Q

How do you choose between using proteolytic digestion or HIER antigen retrieval?

A

Some antigens respond better to protease and some respond better to HIER

Some antigens will need both methods of retrieval

When validating an antibody it is vital we try all combinations of antigen retrieval

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9
Q

Why is antigen retrieval needed

A

We need to undo the cross links created by formalin in order for antibodies to bind

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10
Q

What does it mean to block with hydrogen peroxide, and why is it needed?
(3)

A

Using hydrogen peroxide to quench peroxidase enzyme activity present in blood cells

If this is not quenched it means that red blood cells will stain when you add the DAB

If it’s not done there will be background staining and the red blood cells in the section will stain brown

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11
Q

Why is normal/horse serum needed in immunohistochemistry and how is it used?
(4)

A

Serum is applied before adding the primary antibody so that non specific protein adsorption and binding is blocked

Serum carries antibodies that bind to reactive sites and prevent non specific binding of the secondary antibody

You should use the serum from the source species of the secondary antibody not the primary antibody

Also contains albumin which readily binds to non specific protein binding sites within the sample

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12
Q

Why should you use the serum from the source species of the secondary antibody not the primary antibody?
(2)

A

If you used the primary antibody it would bind to the site that you are interested in labelling

It is used to block secondary antibody binding

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13
Q

What is HEPO

A

Horseradish peroxidase enzyme

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14
Q

what is the difference between indirect and direct IHC?

A

Indirect involves labelling secondary antibody

Direct involves labelling first antibody

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15
Q

What are the three types of labels used in IHC

A

Dyes
Enzymes
Metals

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16
Q

Describe how IHC works
(2)

A

First antibody is anti-human antigen (target) e.g. mouse anti-human

Secondary antibody is from another organism and must be anti-primary antibody e.g. horse anti-mouse

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17
Q

Write about automated immunostaining

A

Rapid, use polymer based immunoperoxidase staining

Automated systems from:
- Roche Ventana
- Dako
- Leica

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18
Q

Describe the immunostaining of epithelial tissues
(2)

A

Cytokeratin can be used for epithelial cell clusters

Chromogranin can be used for endocrine cells

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19
Q

What kinds of antigens can IHC detect

A

Intracellular
Extracellular
Membranous
Nuclear components

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20
Q

Write about mouse and rabbit antibodies used in IHC

A

The animal is injected with purified antigen to stimulate the production of anti-human antigen antibodies

Two types of antibodies can be harvested: Polyclonal or monoclonal

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21
Q

What are polyclonal antibodies

A

Serum containing multiple antibodies to the antigenic epitopes

22
Q

What are monoclonal antibodies

A

The antibody producing cells are harvested from the spleen of the animal, fused with tumour cells and grown in cell culture

grown using hybridoma technology

Highly specific for proteins

23
Q

What does the primary antibody do

A

Binds to epitope(s) of antigen being evaluated

24
Q

What does the secondary antibody do

A

Links to primary antibody and label

25
What type of antibody is used in IHC
IgG is the most common used antibody
26
Give five examples of primary antibodies in cellular pathology
Mouse anti-human macrophage Mouse anti-human smooth muscle actin Mouse anti-human cyclin Rabbit anti-human collagen Rabbit anti-human epithelial cytokeratin
27
Give two examples of secondary antibodies
Swine anti-rabbit igG Rabbit anti-mouse igG
28
Write about the primary antibodies used in IHC (5)
They can be either monoclonal or polyclonal Monoclonal are more specific as they have only one binding site Polyclonal antibodies can bind to several epitopes on the cell of interest resulting in a greater signal and greater chance of staining but it may not be the correct protein that is stained Massive amounts of antibodies are available Need to be validated
29
Write about the secondary antibodies used in IHC (5)
This is the link which binds to the primary antibody and the ABC complex The antibody is biotinylated It increases signal amplification as more than one secondary antibody molecule binds to each primary antibody Should be directed against the species in which the primary antibody was raised Can be labelled with enzyme e.g. peroxidase, fluorochrome labelled or biotinylated
30
What are some detection methods for immunostaining (5)
Applying layers of antibodies and label is more sensitive for antigen detection Direct method - 1 step, label is on primary antibody, it is expensive and there is high background staining In most tissue staining - two or three step methods are preferred e.g. two step polymer-enzyme methods e.g. avidin-biotin-enzyme or fluorochrome methods
31
Give two biotin complexes
Avidin-biotin complex (ABC) method Labelled streptavidin-biotin
32
What is the avidin-biotin complex method
Biotinylated secondary antibodies Avidin biotin reporter enzyme complex
33
What is the labelled streptavidin-biotin method (3)
Variant of the ABC method Uses streptavidin instead of avidin Less non specific binding
34
Write about non biotin polymer based methods (3)
Early polymer methods used a dextran backbone to which multiple enzyme molecules and secondary antibodies are attached Uses a smaller detection complex with less tendency to aggregate Greater sensitivity through better tissue penetration and reduced background staining from endogenous biotin
35
How do we visualise the antigen-antibody complex (6)
Labels are very readily attached to antibodies Antibody labels may be radioactive isotopes, enzymes, biotin, metals, oligonucleotides The label is used to detect the antigenic site where the antibody is binding In cells and tissue immunostaining the label is used to create a colour at site of staining - in situ Commonly a fluorescent dye or enzyme/chromogen label The enzyme catalyses a chemical reaction to produce a colour
36
Give some examples of different types of labels (5)
Radioactive isotopes Enzymes Biotin Metals Oligonucleotides
37
Why does DAB/Peroxidase need to be applied? (3)
Chromogenic detection methods in IHC rely on enzymes that convert soluble substrates into insoluble, chromogenic products Typically conjugated to secondary antibodies e.g. Horseradish peroxidase or alkaline phosphatase Chromogenic detection is usually more sensitive than fluorescent detection
38
How does Horseradish peroxidase work?
It converts 3'3'-diaminobenzidine (DAB) into a brown product
39
How does Alkaline phosphatase work
Alkaline phosphatase (AP) which converts 3-amino-9-ethylcarbozole (ACE) into a real product
40
Explain in your own words how the ABC complex work
Avidin has 4 binding sites 3 out of 4 will be occupied with biotin already 1 of the 4 will bind to biotin on the secondary antibody Once this is bound Avidin molecule has a peroxidase enzyme We have peroxidase in our rbcs -> this is why we block it Biotin is used to just bind the ABC complex
41
Write about the DAB reaction (4)
Add hydrogen peroxide to DAB -> this is where we get the colour Peroxidase enzyme works on hydrogen peroxidase -> Oxygen and H2O released Diaminobenzidene is the chromogen DAB polymer formed (Brown colour results)
42
Explain how the DAB chromogen is formed
Hydrogen peroxidase is the substrate Peroxidase enzyme is linked through antibody to antigen Oxygen and H2O released to form DAB
43
Give an example of a DAB reactoin
Staining for oncogene Brown staining nuclei
44
Give some examples of fluorescent dyes
Fluorescin Lucifer yellow Texas red
45
Give some examples of metal labels
Gold Ferritin (any electron dense metal)
46
Give some examples of metal labels
Gold Ferritin (any electron dense metal)
47
Write about enzyme labels (4)
Peroxidase commonly used -> immunoperoxidase Peroxidase is attached at site of antigen Peroxidase acts on a hydrogen peroxide substrate to release oxygen which causes a brown polymer of diaminobenzidene to form resulting in a brown deposit at site of antigen Other enzyme label - alkaline phosphatase: use Azo dye reaction to visualise (red)
48
Write about the use of haematoxylin dye (4)
Mayers haematoxylin used as a counterstain Needs to be blued so rinse in running water to increase the pH Nucleus turns from purple to a blue/violet colour Other counterstains can be used but are rarely part of kits e.g. nuclear fast red or methyl green
49
Why do we use positive and negative controls
Positive controls indicate the test has worked Negative controls indicate that there is no background or non specific staining- does not get primary antibody
50
Give an example of where immunoperoxidase is used
Oestrogen receptor in breast tumour cells
51
Give an example of where immunoalkaline phosphatase is used
Its a red chromogen - cytoplasmic stain Tumour cells in clusters

2.2 Cellular Pathology (29 decks)

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