Fixation and blocking for immunofluorescence

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PFA fixates the cells by crosslinking (covalent binding) specific amino acids. Why does this need to happen before the immunostaining?

Pick one

1. Antibodies only bind to fixated proteins by binding to the crosslinks.
2. Antibodies are broken down by living cells, so they must be fixated first.
3. Cell fixation is needed to stop all protein synthesis and degradation at the same time point. (Correct)
4. All the above statements are true.

PFA is not the only chemical that is added to the cells before immunostaining. Triton is also used, but what is the function of Triton?
Pick one

1. Triton makes small holes in the cells so that the antibody can enter the cell.
2. Triton is a catalysator for the fixation procedure.
3. PFA is a solid matter and Triton is the liquid to dissolve it in
4. Because of the Triton, the cells will not be stressed during fixation.

Why do you need to add blocking proteins before antibody staining?
Choose one or more options:

1. To enhance background signal in the detection of the IF final product.
2. To prevent the occurrence of false positives.
3. To prevent the interaction between the antibody and the unspecific binding to the overall present proteins instead of binding to the target protein solely.
4. To treat, i.e. ‘prepare’, the cells so that they can bind antibodies.

Which methods can be used to block samples prior to antibody staining?
Choose one or more options:

1. Non-fat dry milk (correct)
2. PBS-0.5% BSA solution (correct)
3. Paraformaldehyde (PFA) in PBS
4. I-Block with 0.1% of detergent (Tween 20 or Triton X-100) (correct)
5. I-Block with 50% of detergent (Tween 20 or Triton X-100)

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