SIMPLE: a sequential immunoperoxidase labeling and erasing method.
The ability to simultaneously visualize expression of multiple antigens in cells and tissues can provide powerful insights into cellular and organismal biology. However, standard methods are limited to the use of just two or three simultaneous probes and have not been widely adopted for routine use in paraffin-embedded tissue. We have developed a novel approach called sequential immunoperoxidase labeling and erasing (SIMPLE) that enables the simultaneous visualization of at least five markers within a single tissue section. Utilizing the alcohol-soluble peroxidase substrate 3-amino-9-ethylcarbazole, combined with a rapid non-destructive method for antibody-antigen dissociation, we demonstrate the ability to erase the results of a single immunohistochemical stain while preserving tissue antigenicity for repeated rounds of labeling. SIMPLE is greatly facilitated by the use of a whole-slide scanner, which can capture the results of each sequential stain without any information loss.
Glass
George
G
University of Virginia, UVa Health System, Charlottesville, VA 22908, USA.
Papin
Jason A
JA
Mandell
James W
JW
D000368:Aged; D000818:Animals; D000906:Antibodies; D015415:Biomarkers / Q000378:metabolism; D001921:Brain / Q000378:metabolism; D064026:Calbindins; D002227:Carbazoles; D004396:Coloring Agents; D005904:Glial Fibrillary Acidic Protein; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000276:immunology / Q000378:metabolism; D009414:Nerve Growth Factors / Q000276:immunology / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000276:immunology / Q000378:metabolism; D016900:Neurofilament Proteins / Q000276:immunology / Q000378:metabolism; D010902:Pituitary Gland / Q000378:metabolism; D064030:S100 Calcium Binding Protein G / Q000276:immunology / Q000378:metabolism; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000276:immunology / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods
D000906:Antibodies; D015415:Biomarkers; D064026:Calbindins; D002227:Carbazoles; D004396:Coloring Agents; D005904:Glial Fibrillary Acidic Protein; D008869:Microtubule-Associated Proteins; C079240:Mtap2 protein, mouse; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D016900:Neurofilament Proteins; D064030:S100 Calcium Binding Protein G; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; C528900:glial fibrillary astrocytic protein, mouse; C061195:neurofilament protein M; C020702:3-amino-9-ethylcarbazole