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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
Q

What type of antibody is used in IHC

A

IgG is the most common used antibody

26
Q

Give five examples of primary antibodies in cellular pathology

A

Mouse anti-human macrophage

Mouse anti-human smooth muscle actin

Mouse anti-human cyclin

Rabbit anti-human collagen

Rabbit anti-human epithelial cytokeratin

27
Q

Give two examples of secondary antibodies

A

Swine anti-rabbit igG
Rabbit anti-mouse igG

28
Q

Write about the primary antibodies used in IHC
(5)

A

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
Q

Write about the secondary antibodies used in IHC
(5)

A

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
Q

What are some detection methods for immunostaining
(5)

A

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
Q

Give two biotin complexes

A

Avidin-biotin complex (ABC) method

Labelled streptavidin-biotin

32
Q

What is the avidin-biotin complex method

A

Biotinylated secondary antibodies

Avidin biotin reporter enzyme complex

33
Q

What is the labelled streptavidin-biotin method
(3)

A

Variant of the ABC method

Uses streptavidin instead of avidin

Less non specific binding

34
Q

Write about non biotin polymer based methods
(3)

A

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
Q

How do we visualise the antigen-antibody complex
(6)

A

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
Q

Give some examples of different types of labels
(5)

A

Radioactive isotopes

Enzymes

Biotin

Metals

Oligonucleotides

37
Q

Why does DAB/Peroxidase need to be applied?
(3)

A

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
Q

How does Horseradish peroxidase work?

A

It converts 3’3’-diaminobenzidine (DAB) into a brown product

39
Q

How does Alkaline phosphatase work

A

Alkaline phosphatase (AP) which converts 3-amino-9-ethylcarbozole (ACE) into a real product

40
Q

Explain in your own words how the ABC complex work

A

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
Q

Write about the DAB reaction
(4)

A

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
Q

Explain how the DAB chromogen is formed

A

Hydrogen peroxidase is the substrate

Peroxidase enzyme is linked through antibody to antigen

Oxygen and H2O released to form DAB

43
Q

Give an example of a DAB reactoin

A

Staining for oncogene

Brown staining nuclei

44
Q

Give some examples of fluorescent dyes

A

Fluorescin
Lucifer yellow
Texas red

45
Q

Give some examples of metal labels

A

Gold
Ferritin
(any electron dense metal)

46
Q

Give some examples of metal labels

A

Gold
Ferritin
(any electron dense metal)

47
Q

Write about enzyme labels
(4)

A

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
Q

Write about the use of haematoxylin dye
(4)

A

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
Q

Why do we use positive and negative controls

A

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
Q

Give an example of where immunoperoxidase is used

A

Oestrogen receptor in breast tumour cells

51
Q

Give an example of where immunoalkaline phosphatase is used

A

Its a red chromogen - cytoplasmic stain

Tumour cells in clusters

2.2 Cellular Pathology (29 decks)

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