IF, IHC and ICC

Question 1

What does IF stand for?

Answer 1

Immunofluorescence

Question 2

What does IHC stand for?

Answer 2

Immunohistochemistry

Question 3

What does ICC stand for?

Answer 3

Immunocytochemistry

Question 4

Where is the variable region of an antibody? Top or bottom?

Answer 4

Top

Question 5

What does the antibody bind to?

Answer 5

Epitope on the antigen

Question 6

How many types of mammalian immunoglobulin chains are there?

Answer 6

5
IgA, IgD, IgE, IgG and IgM

Question 7

What are the three sections an antibody can be divided into?

Answer 7

Two Fab regions
One Fc region

Question 8

Which region of the antibody is the variable domain in?

Answer 8

Fab regions

Question 9

What binds to the Fc fragment?

Answer 9

• Fc receptors on lymphocytes
• Secondary antibodies
• Dye labels and enzymes

Question 10

What enzyme do we use if we want to produce a fragment both Fab regions and no Fc?

Answer 10

Pepsin

Question 11

What enzyme do we use if we want to produce all three parts?

Answer 11

Papain

Question 12

Which antibody is widely produced, found in mucosal areas and prevents colonisation of pathogens?

Answer 12

IgA

Question 13

Which antibody is found with IgM on the surface of B cells? And is involved in B cell development?

Answer 13

IgD

Question 14

Which antibody is important in allergies and binds to allergens causing histamine release?

Answer 14

IgE

Question 15

Which is the major antibody and produces the most antibody-based immunity against invading pathogens?

Answer 15

IgG

Question 16

Which is the first response antibody, has high avidity (strength) and eliminates pathogens in early B cell immunity? (before sufficient IgG?)

Answer 16

IgM

Question 17

Which antibody type is a mixture of many types of antibodies?

Answer 17

Polyclonal antibody

Question 18

Why are multiple antibodies produced in response to an antigen?

Answer 18

Because antigens have multiple epitopes.

Question 19

From where in the animal’s body are antibodies collected?

Answer 19

Antiserum (whole serum following exposure to antigen).

Question 20

Why are lab animals injected twice with the same antigen?

Answer 20

2nd infection activates memory cells to make IgG antibodies.

Question 21

What is affinity maturation?

Answer 21

Affinity maturation is the process by which B cells undergo selection to produce antibodies with increased binding affinity for a specific antigen.

Leads to a more effective immune response upon re-exposure.

Question 22

What is an adjuvant?

Answer 22

Adjuvant is a substance that is added to a vaccine to enhance the body’s immune response to the vaccine’s antigen.

Stimulates greater antibody production

Question 23

Monoclonal antibody production:
What is collected from the lab animal after infecting it? And where in the body it collected from?

Answer 23

B cells
Collected from spleen of lab animal

Question 24

What are collected B cells fused with in monoclonal antibody production?

Answer 24

B cells fused with immortal cancerous B cells (myeloma cells).

Question 25

Why are B cells fused with immortal cancerous B cells?

Answer 25

To yield hybridoma cells that can proliferate and produce antibodies continuously.

Question 26

What are hybridoma cells screened for?

Answer 26

Desired monoclonal antibody (recognising single epitope).

Question 27

How do we develop a secondary antibody?

Answer 27

Immunise a second animal with IgG from mouse. Animal produces IgG in response to constant region of mouse IgG.

Question 28

What is immunofluorescence?

Answer 28

Visualises presence and location of specific proteins/ antigens in cells/tissues using antibodies labeled with fluorescent dyes

Question 29

Can Immunofluorescence also be used to detect protein modifications?

Answer 29

Yes

Question 30

What are the two types of Immunofluorescence (IF)?

Answer 30

1. Direct 2. Indirect

Question 31

What is the purpose of the fixation step in Immunofluorescence (IF)?

Answer 31

To preserve original cell architecture and ensuring antigens are immobilised.

Question 32

What is the purpose of organic solvents such as methanol and acetone in the fixation step of IF?

Answer 32

Organic solvents remove lipids from the cell membrane, facilitating antibody binding. Acetone is more harsh.

Question 33

What types of aldehyde are used in aldehyde based fixing? What is the purpose of aldehyde-based fixing?

Answer 33

4% formaldehyde or 10% formalin To cross-link proteins.

Question 34

Why is permeabilisation step in IF needed?

Answer 34

Allows antibodies to access intracellular antigens within cells. Creates openings in membrane.

Question 35

If we used organic solvent in fixation step, do we need to permeate?

Answer 35

No, as cell membrane holes are already punched.

Question 36

If aldehyde fixative used, do we permeate? If so, what do we use to do so?

Answer 36

Yes. Non-ionic detergent such as Triton X-100 or saponin.

Question 37

How do we reduce non-specific antibody binding during IF?

Answer 37

Blocking

Question 38

What are two commonly used blocking reagents in IF?

Answer 38

1% milk powder in PBS Serum from the 2nd animal Both bind unoccupied sites

Question 39

What is the function of the microtone during IF and IHC?

Answer 39

To create uniform sections of tissue samples of a specified thickness.

Question 40

How does ImmunoHistoChemistry (IHC) differ from Immunofluorescence (IF)?

Answer 40

secondary antibody is enzyme-linked in IHC

Question 41

How does ImmunoCytoChemsitry (ICC) differ from ImmunoHistoChemsitry (IHC)?

Answer 41

ICC involves staining cells (with secondary antibodies), not tissue.