Immunohistochemistry Flashcards
What is immunocyto/histochemistry
An indirect staining method using labelled antibodies to detect specific antigens/epitopes in cells/tissues
What are four characteristics of immunostaining
In situ -> shows the location of the component in cell/tissue
Specific
Sensitive
Valuable in tumour typing, immunology and research
How does immunostaining work?
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
What must be done prior to immunostaining
Antigen retrieval
What are the two main antigen retrieval methods?
Proteolytic digestion
Heat induced epitope retrieval (HIER)
What is meant by proteolytic digestion for antigen retrieval
This is where tissue sections are incubated with an enzyme e.g. trypsin or protease
What is meant by HIER antigen retrieval
HIER uses either a pressure cooker or a microwave
Slides are placed in a buffer of either pH6 or pH9 and heated
How do you choose between using proteolytic digestion or HIER antigen retrieval?
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
Why is antigen retrieval needed
We need to undo the cross links created by formalin in order for antibodies to bind
What does it mean to block with hydrogen peroxide, and why is it needed?
(3)
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
Why is normal/horse serum needed in immunohistochemistry and how is it used?
(4)
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
Why should you use the serum from the source species of the secondary antibody not the primary antibody?
(2)
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
What is HEPO
Horseradish peroxidase enzyme
what is the difference between indirect and direct IHC?
Indirect involves labelling secondary antibody
Direct involves labelling first antibody
What are the three types of labels used in IHC
Dyes
Enzymes
Metals
Describe how IHC works
(2)
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
Write about automated immunostaining
Rapid, use polymer based immunoperoxidase staining
Automated systems from:
- Roche Ventana
- Dako
- Leica
Describe the immunostaining of epithelial tissues
(2)
Cytokeratin can be used for epithelial cell clusters
Chromogranin can be used for endocrine cells
What kinds of antigens can IHC detect
Intracellular
Extracellular
Membranous
Nuclear components
Write about mouse and rabbit antibodies used in IHC
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
What are polyclonal antibodies
Serum containing multiple antibodies to the antigenic epitopes
What are monoclonal antibodies
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
What does the primary antibody do
Binds to epitope(s) of antigen being evaluated
What does the secondary antibody do
Links to primary antibody and label
What type of antibody is used in IHC
IgG is the most common used antibody
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
Give two examples of secondary antibodies
Swine anti-rabbit igG
Rabbit anti-mouse igG
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
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
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
Give two biotin complexes
Avidin-biotin complex (ABC) method
Labelled streptavidin-biotin
What is the avidin-biotin complex method
Biotinylated secondary antibodies
Avidin biotin reporter enzyme complex
What is the labelled streptavidin-biotin method
(3)
Variant of the ABC method
Uses streptavidin instead of avidin
Less non specific binding
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
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
Give some examples of different types of labels
(5)
Radioactive isotopes
Enzymes
Biotin
Metals
Oligonucleotides
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
How does Horseradish peroxidase work?
It converts 3’3’-diaminobenzidine (DAB) into a brown product
How does Alkaline phosphatase work
Alkaline phosphatase (AP) which converts 3-amino-9-ethylcarbozole (ACE) into a real product
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
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)
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
Give an example of a DAB reactoin
Staining for oncogene
Brown staining nuclei
Give some examples of fluorescent dyes
Fluorescin
Lucifer yellow
Texas red
Give some examples of metal labels
Gold
Ferritin
(any electron dense metal)
Give some examples of metal labels
Gold
Ferritin
(any electron dense metal)
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)
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
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
Give an example of where immunoperoxidase is used
Oestrogen receptor in breast tumour cells
Give an example of where immunoalkaline phosphatase is used
Its a red chromogen - cytoplasmic stain
Tumour cells in clusters
