Question

1. What would you see if you forgot to add the primary antibody? If you forgot the secondary antibody? If the blocking agent didn’t work? If you did not permeabilize your cells correctly?

Answer 1

Immunofluorescence(IF) is a powerfu method for visualising intracellular processes,conditions andd structures.

Two variants are there,direct and indirect IF.

In direct or primary IF a specific primary antibody is linked to a fluorochrome, is used for binding to the target structure and its direct visualisation.In Indirect or secondary IF,a two step incubation is performed.Firstly a specific primary antibody recognizes the Target structure.Then a fluorochrome coupled secondary antibody is applied which specifically binds to the primary antibody and visualises the target structure.

A primary antibody is an immunoglobulin that specifically binds to a particular protein or other biomolecule of research interest for the purpose of purifying or detecting and measuring it.If primary antibody is not added,non specific binding or false positives may result due to non specific binding of the secondary antibody.

A secondary antibody aids in the detection,sorting or purification of target antigens by binding to the primary antibody,which directly binds to the target antigen.Secondary antibodies bind to the heavy chains of primary antibodies,so that they dont interfere with the primary antibody binding to the antigen.so they are for indirect detection of a specific target.Using secondary antibody gives a greater flexibibility in choosing antibodies and fluorochromes and furthermore to a signal amplification,because several secondary antibody molecules can bind to one primart antibody.

Blocking ia an important step in IF for minimising unspecific binding of the primary antibody within the cell.Protiens from bovine serum albumin, milk powder or serum can be used.These blocking proteins do not originate from the species in which the primary antibody was raised,otherwise the specificity of secondary antibody to primary antibody will be lost.If blocking agent did not work unspecific binding of antibodies to non target structures resulting in false positive signals within the cell takes place.

By permeabilisation,intracellular structures become accessible for antibodies which are otherwise unable to pass through the lipid membranes of the cell.A separate permeabilisation step is necessary,depending on type of fixation.Detergents are mostly used for permeabilisation.if cells are not permeablised correctly ,antibodies fail to cross the lipid membrane of the cell and fail to reach the target.