Method for improved cell identification

A method of visualizing a plurality of cell types in a microscopic multicell tissue sample comprising cells of at least one particular cell type as well as cells of at least one confounding cell type, wherein cells of said at least one particular cell type and cells of said at least one confounding cell type both comprise a second cell marker and cells of the at least one confounding cell type also comprise a first cell marker not present in cells of the at least one particular cell type, comprising the steps of:

• a) providing a microscopic multicell tissue sample known to comprise cells of at least one confounding cell type as well as cells of at least one particular cell type;
• b) incubating the microscopic multicell tissue sample of step a) with a first cocktail of at least one antibody-like ligand, which antibody-like ligand(s) of the cocktail (is)are capable of binding specifically to cell marker(s) on confounding cells but not to cell markers on cells of said at least one particular cell type, and where the sample is incubated with the first cocktail under conditions favourable for formation of specific bonds between said cell markers on confounding cells and said ligands, resulting in formation of complexes between said cell markers of said cells and said ligands;
• c) incubating the complexes formed in step b) with a first detecting antibody-like ligand preparation, the first detecting antibody-like ligand being conjugated to a first detection enzyme, said first detecting antibody-like ligand being capable of binding specifically to the antibody-like ligands of said first cocktail, where the incubation is carried out under conditions favourable for formation of specific bonds between said first detecting antibody-like ligand and the antibody-like ligand(s) of said first cocktail, resulting in formation of complexes between said first detecting antibody-like ligand and the complexes formed in step b);
• d) adding a first substrate preparation comprising a substrate for said first detection enzyme to the complex formed in step c) and incubating the resulting mixture under conditions that are favorable for formation of a detectable polymer thereby obtaining detectable accumulated polymers inside or in the vicinity of confounding cells of said microscopic multicell tissue sample;
• e) adding a first primary antibody-like ligand preparation, wherein the ligand is capable of binding specifically to a cell marker on said particular cell type, and incubating the resulting mixture under conditions favourable for formation of complexes between said cell marker and said first primary antibody-like ligand;
• f) adding a first secondary antibody-like ligand preparation to the resulting mixture of step e), the first secondary antibody-like ligand being conjugated to a fluorochrome, said first secondary antibody-like ligand being capable of binding specifically to said first primary antibody-like ligand, and incubating the resulting mixture under conditions favourable for formation of specific bonds between said first primary and first secondary antibody-like ligands;
• g) removing unbound secondary antibody-like ligands, for instance through a washing step;
• h) chemically fixating complexes of bound primary and secondary antibody-like ligands;
• i) exposing cells of the resulting sample to a group of at least one fluorochrome-labelled antibody-like ligand preparation(s), each preparation of said group of at least one fluorochrome-labelled antibody-like ligand preparation(s) being capable of specifically binding to a unique cell marker associated with a particular cell type of interest, each preparation of said group of at least one fluorochrome-labelled antibody-like ligand preparation(s) having a unique fluorochrome capable of sending out a unique fluorescence signal;
• j) removing unbound fluorochrome-labelled ligands, for example through a washing step;
• and
• k) during the course of the method, visualizing said at least one particular cell type and said at least one confounding cell type by detecting fluorescence signals obtained from one or more fluorochrome(s), and by detecting the blocking polymer that has been formed around said at least one confounding cell type.