title: Chromogen-based immunohistochemical method for elucidation of the coexpression of two antigens using antibodies from the same species.
abstract: The authors established a chromogen-based, double immunolabeling method using antibodies from the same species without any unwanted cross-reactivity. In addition, time-consuming staining steps were shortened by using polymer-based secondary antibodies. Taking advantage of the nature of the chromogen 3-amino-9-ethylcarbazole (AEC), which is used as a horseradish peroxidase substrate for antibody detection, the AEC-derived signals in the first color development were easily eliminated by alcohol treatment. Therefore, the signals from the first staining did not interfere with those from the subsequent second staining, which used the chromogen 3,3'-diaminobenzidine. The co-localization of antigens within the same cell could be confirmed using this method, because cell images of the individual dye staining steps could be obtained and developed. The images from each step could be expressed in pseudo-colors in a dark field by using a computer. As a result, merged images could be constructed that resembled the images acquired by the fluorescent immunolabeling technique. The resolution of this method enabled analysis of the coexpression of two antigens in the same cell in the same section. The authors have named this staining technique the elucidation of the coexpression of two antigens in a cell using antibodies from the same species (ECSS).
authors.affiliation: Department of Health Science, Ishikawa Prefectural Nursing University, Kahoku, Ishikawa, Japan. tnakata1@mail.goo.ne.jp
authors.forename: Takahiro
authors.initials: T
authors.lastname: Nakata
authors.forename: Natsuki
authors.initials: N
authors.lastname: Suzuki
mesh_terms: D015100:3,3'-Diaminobenzidine / Q000737:chemistry*; D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D000941:Antigens / Q000276:immunology / Q000378:metabolism*; D034961:Antigens, Nuclear / Q000276:immunology / Q000378:metabolism; D002227:Carbazoles / Q000737:chemistry*; D002863:Chromogenic Compounds / Q000737:chemistry*; D003429:Cross Reactions; D005904:Glial Fibrillary Acidic Protein / Q000276:immunology / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000737:chemistry; D007150:Immunohistochemistry / Q000379:methods*; D008297:Male; D051379:Mice; D009419:Nerve Tissue Proteins / Q000276:immunology / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D013116:Spinal Cord / Q000378:metabolism; D016708:Synaptophysin; D050494:Vesicular Acetylcholine Transport Proteins / Q000276:immunology / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000276:immunology / Q000378:metabolism
chemical_list: D000906:Antibodies; D000941:Antigens; D034961:Antigens, Nuclear; D002227:Carbazoles; D002863:Chromogenic Compounds; D005904:Glial Fibrillary Acidic Protein; D009419:Nerve Tissue Proteins; C546339:Rbfox3 protein, rat; C493461:Slc18a3 protein, rat; D016708:Synaptophysin; C569016:Syp protein, rat; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins; D015100:3,3'-Diaminobenzidine; C020702:3-amino-9-ethylcarbazole; D006735:Horseradish Peroxidase