In a second incubation step the fluorescence-coupled secondary antibody is applied which binds to the first antibody and therefore visualizes the target structure. Next the incubation with the first antibody takes place, which specifically recognizes epitopes on the target molecule. Blocking with normal serum, milk powder or bovine serum albumin reduces the unspecific binding of antibodies to non-target structures in order to minimize false-positive signals. Next a permeabilization step with detergents is performed to enable the antibodies to cross the cellular membranes. After cultivation, cells get fixed and therefore killed with a chemical crosslinker (e.g. 1: This chart illustrates a typical workflow of indirect Immunofluorescence (IF) with epithelial cells adherently growing on coverslips.
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