Improved catalyzed reporter deposition, iCARD.
Novel reporters have been synthesized with extended hydrophilic linkers that in combination with polymerizing cross-linkers result in very efficient reporter deposition. By utilizing antibodies to stain HER2 proteins in a cell line model it is demonstrated that the method is highly specific and sensitive with virtually no background. The detection of HER2 proteins in tissue was used to visualize individual antigens as small dots visible in a microscope. Image analysis-assisted counting of fluorescent or colored dots allowed assessment of relative protein levels in tissue. Taken together, we have developed novel reporters that improve the CARD method allowing highly sensitive in situ detection of proteins in tissue. Our findings suggest that in situ protein quantification in biological samples can be performed by object recognition and enumeration of dots, rather than intensity-based fluorescent or colorimetric assays.
Dako A/S , Produktionsvej 42, 2600 Glostrup, Denmark.
Jesper
J
Lohse
Kenneth Heesche
KH
Petersen
Nina Claire
NC
Woller
Hans Christian
HC
Pedersen
Galina
G
Skladtchikova
Rikke Malene
RM
Jørgensen
D000906:Antibodies / Q000737:chemistry / Q000378:metabolism*; D001560:Benzidines / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D045744:Cell Line, Tumor; D003432:Cross-Linking Reagents / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D015394:Molecular Structure; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis* / Q000096:biosynthesis
D000906:Antibodies; D001560:Benzidines; D003432:Cross-Linking Reagents; D006735:Horseradish Peroxidase; D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human