Lecture 2

Question 1

How do you label molecules in cells?

Answer 1

1) immunofluorescence
2) immunogold
3) making antibodies
4) microinjection
5) green fluorescent protein

Question 2

What are the steps of indirect immunofluorescence?

Answer 2

1) live cell (antibodies cant cross membrane)
2) formaldehyde fix – kills cell
3) detergent – permeabilize cell
4) add primary antibody
5) add secondary antibody

Question 3

compare indirect vs direct immunofluorescence

Question 4

what are the important variations on immunofluorescence?

Answer 4

1) fix/permeabilize with methanol
2) skip permeabilization step (stain surface only)
3) Use small amount of gluteraldehyde for
stronger fixation: disadvantage is that it is
fluorescent
4) Tissues are usually sectioned prior to staining
(unlike cells in tissue culture)

Question 5

What are the advantages/disadvantages for using antibodies together for multiple fluorescent labels?

Answer 5

1) track multiple proteins at once
2) up to 3 colours can be used together, sometimes 5 or more
BUT sometimes dk the localization of 1 protein -> compare it to known proteins in the same cell

Question 6

What are antibodies?

Answer 6

= immunoglobulins produced by B-lymphocytes and plasma cells of vertebrates

= Y-shaped molecules with
- 2 light
- 2 heavy chains
- 2 antigen-binding sites, one at the tip of each arm of the Y

Question 7

What are the most commonly used antibodies in immunocytochemistry?

Answer 7

IgG class of immunoglobulins