Immunohistochemistry and Immunocytochemistry

Question 1

Why are histological dyes e.g. Haemtoxylin and Eosin used?

Answer 1

To identify subcellular structures found within cells.

Histological dyes are needed because cells are transparent, therefore it can be difficult to see subcellular structures.

Question 2

What is immunohistochemistry?

Answer 2

Immunohistochemistry is the application of antibodies to identify subcellular structurs within a tissue/ cell.

For example, microtubules can be specifically identified using tubulin specific antibodies.

Question 3

What is confocal flourescent microscopy?

Answer 3

Confocal flourescent microscopy is a microscopic technique used to visualise structures in 3D.

The image becomes processed by a computer.

Question 4

Where is confocal microscopy used?

Answer 4

Confocal microscopy is often used when dealing with thick tissue samples.

Question 5

Do you use confocal flourescent microscopy for flat strucutres within cells?

Answer 5

No, wide-field microscopes are used instead to visualise flat cellular components withn cells.

Question 6

Which type of cells produce antibodies?

Answer 6

B lymphocytes produce antibodies.

Question 7

What happens to B cells when it binds to antigens?

Answer 7

When antigens bind to surface IgM, B cells begin to proliferate rapidly - this is known as clonal expansion.

These cells then begin to start producing soluble antibodies that are specific to the antigen being processed.

Question 8

Describe the structure of an antibody?

Answer 8

The antibody has the following structural components:
> Variable region ==> this is where the antigen will bind - it is specific to each antigen type.
> Constant region (Fc fragment) - this is the same for all antibodies of the same species.

Remember the heavy chain determines the class.Different classes possess different functions.

Question 9

How do we use antibodies to see cells or their components?

Answer 9

We can see cellular components in 2 ways:

1) One way to see cellular components is by linking the antibody to an enzyme (e.g. alkaline phosphatase). When the substrate becomes added, a coloured reaction product marks the presence of the antigen.

The amount of antibody bound is equivalent to the amount of antigen present.

2) Another way to visualise cellular components is by uising fluorophores.
The binding is detected by using a flourescent microscope.

Question 10

What are some common examples of flourophores being used at the moment?

Answer 10

Common flourophores used  for immunohistochemistry  includes: 
> TRITC 
> Rhodamine 
> FITC 
> Flourescein 
> Cy3
> Cy5
> Alexa

Question 11

Antibodies that recognise their target antigen of interest are called what?

Answer 11

Primary antibodies

Question 12

Primary antibodies can raised in different species of animals most commonly in which?

Answer 12

> Mice
Rabbits
Goats
Sheep

Question 13

What are the 2 classes of primary antibodies?

Answer 13

1) Monoclonal antibodies

2) Polyclonal antibodies

Question 14

What are monoclonal antibodies?

Answer 14

Monoclonal antibodies are antibodies derived from a single clone of B cells. All of the antibodies are therefore identical and therefore recognise only one specific site on the antigen.

Question 15

What are polyclonal antibodies?

Answer 15

Polyclonal antibodies are antibodies derived from several B cell clones.

Polyclonal antibodies are a mixture of antibodies that all recognise the same antigen, but different target sites on it.

Question 16

Monoclonal antibodies are usually raised in which type of animals? What is an example of a monoclonal antibody that has been raised in this type of animal?

Answer 16

Monoclonal antibodies are usually raised in mice.

For example:
Mouse anti-human actin monoclonal antibody

Question 17

What type(s) of animals are polyclonal antibodies raised in?

Answer 17

Polyclonal antibodies are usually raised in rabbits, goats or sheep.

Question 18

What is an example of a polyclonal antibody that has been raised in these animal types?

Answer 18

Sheep anti-human actin polyclonal antibody - is a mixture of antibodies raised in sheep injected with human actin protein.

Question 19

What are secondary antibodies?

Answer 19

Antibodies that target Fc fragments of other antibodies.

Question 20

What are examples of secondary antibodies?

Answer 20

Anti-mouse Fc ==. recognises all mouse antibodies.

Anti-rabbit Fc recognises all rabbit antibodies.

Question 21

What is the most versatile way of doing flourescence immunostaning?

Answer 21

The most versatile way of doing flourescence immunostaining is by using flourscently-conjugated species specific secondary antibodies that recognise the Fc portion of the primary antibody, rather than directly coupling each primary antibody to a flourophore.

Thus, it is really important to note the species in which the secondary antibodies are raised in.

Question 22

When raiisng secondary antibodies, what other factors do you need to take into consideration?

Answer 22

You also need to take into account the isotype of the antibody you are raising e.g. if it is IgM or IgG.

In an immune response, the majority of the antibodies raised in response to an antigen are IgG, so polyclonal antibodies contain mostly IgG isotype antibodies. Secondary antibodies for polyclonal primary antibodies are therefore anti-IgG.

Question 23

How can we amplify the signal further?

Answer 23

To amplify the signal further, we need to use biotinylated secondary antibodies or flourophore-coupled streptavidin which as multiple binding sites for biotin.

Question 24

When are cells usually immunostained?

Answer 24

Cells are usually immunostained after fixing.

Question 25

What is meant by fixing?

Answer 25

Fixing is a process where cells are fixed in the position they were before the fixing chemical was applied.

Question 26

What are commonly used fixatives?

Answer 26

> Paraformaldehyde
Formaldehyde
Glutaraldehyde

Note that some antibodies do not recognise an antigen after it is fixed and as a result, they have to be used on live cells (if the antigen is on the cell surface).

Question 27

Cells can also be stained live. What temperatures do we need to consider?

Answer 27

4 degrees vs RT vs 37 degrees

At 37 degrees or even at RT, cells tend to cluster, patch, cap or even undergo endocytosis i.e. antigen changes its location as a consequence of antibody binding.

At 4 degrees, the antigen is immobile, so the distribution revealed by the antibody would be a true reflection of the reality.

Question 28

When staining cells live, what else do we need to consider?

Answer 28

Buffering - cells need to be buffered appropriateyly - especially if they are at 4 degrees or room temperature - might need to add mM Hepes.

Question 29

What do you have to do if the antigen is inside the cell?

Answer 29

If the antigen is inside the cell, then the cell needs to be permeablised by producing small holes in the cell membrane, allowing free access to cell interior.

Question 30

What reagents are used for cell permeabilisation?

Answer 30

Methanol

Triton-X100 (Detergent 0.2%)

Question 31

Permeabalisation is not compatible with which type of staining?

Answer 31

Permeablisation is not compatible with LIVE staining - this is because the cells would die as a result of permeabilisaiton. Only cell surface antigens can be stained live.

Question 32

What do you have to do for non-specific binding sites?

Answer 32

Non-specific binding sites should be blocked by incubating ces with medium/PBS containing proteins such as :
> foetal calf serum (FCS)
> Bovine serum albmin (e..g 5% )
Without this step, background binding of antibody might be high preventingspecifc binding to target antigens.

Question 33

What is co-immunocytochemistry??

Answer 33

Identifying the relevant distributiion of two or more antigens.

Question 34

How can we carry out co-immunocytochemistry?

Answer 34

Raise primary antibodies in animals of different species or in the same animal but of different isotypes e.g. co-immunocytochemistry can be carried out using two mouse monoclonal antibodies, as long as one is an IgG isotype and the other an IgM isotype.

Different secondary antibodies can be used to recognise different primary antibodies. Each of the secondary antibodies must be coupled with a different flourophore.

Question 35

What is flourescence activated sorting (FACS)?

Answer 35

Utilises immunofluorescence for immunocytochemistry
Single-cell suspension is incubated with fluorophore-labelled antibodies
Unbound Ab are washed
Cell suspension is acquired at FACS machine (acquisition)
FACS data are analysed offline (analysis)