TY - JOUR
T1 - Double-labeling for keratin and class III β-tubulin within cultured retinal pigment epithelial cells
T2 - Comparison of chromogens to yield maximum resolution of two structural proteins within the same cell
AU - Vinores, Stanley A.
AU - Vinores, Melissa A.
AU - Chiu, Charlie
AU - Woerner, Thomas M.
AU - Campochiaro, Peter A.
PY - 1997/1
Y1 - 1997/1
N2 - Previous attempts at colocalization of 2 distinct antigens within the same cell have met with various limitations. Either the double-labeling preparation was not permanent and required a fluorescent microscope (immunofluorescence) or I chromogen would totally or partially obscure the other, making resolution of both markers difficult. The present study was conducted to assess different combinations of chromogens for optimal colocalization of 2 structural proteins, keratin and class III β-tubulin, within cultured retinal pigment epithelial cells. The preferred combination of chromogens was HistoMark Red/HistoMark Blue. Good results were also achieved with HistoMark Red/ 3,3'-diamino-benzidine-HCl (DAB) or True Blue, HistoMark Blue/DAB, 3-amino-9-ethylcarbazole (AEC) or HistoMark Orange, and HistoMark Orange/ HistoMark Black or True Blue. It was unimportant which chromogen was used to localize each protein, but the order in which the chromogens were developed was important since some chromogens would wash out or diminish in clarity through subsequent incubations. The sequence of the chromogen application should be DAB first, then HistoMark Red, HistoMark Blue, AEC or Histo-Mark Orange, HistoMark Black, and lastly, 4-chloro-1- naphthol or True Blue.
AB - Previous attempts at colocalization of 2 distinct antigens within the same cell have met with various limitations. Either the double-labeling preparation was not permanent and required a fluorescent microscope (immunofluorescence) or I chromogen would totally or partially obscure the other, making resolution of both markers difficult. The present study was conducted to assess different combinations of chromogens for optimal colocalization of 2 structural proteins, keratin and class III β-tubulin, within cultured retinal pigment epithelial cells. The preferred combination of chromogens was HistoMark Red/HistoMark Blue. Good results were also achieved with HistoMark Red/ 3,3'-diamino-benzidine-HCl (DAB) or True Blue, HistoMark Blue/DAB, 3-amino-9-ethylcarbazole (AEC) or HistoMark Orange, and HistoMark Orange/ HistoMark Black or True Blue. It was unimportant which chromogen was used to localize each protein, but the order in which the chromogens were developed was important since some chromogens would wash out or diminish in clarity through subsequent incubations. The sequence of the chromogen application should be DAB first, then HistoMark Red, HistoMark Blue, AEC or Histo-Mark Orange, HistoMark Black, and lastly, 4-chloro-1- naphthol or True Blue.
KW - chromogens
KW - class III β-tubulin
KW - double-labeling
KW - epithelial cells
KW - immunohistochemistry
KW - keratin
KW - retinal pigment epithelial cells
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U2 - 10.1179/his.1997.20.1.19
DO - 10.1179/his.1997.20.1.19
M3 - Article
AN - SCOPUS:0030933576
SN - 0147-8885
VL - 20
SP - 19
EP - 26
JO - Journal of Histotechnology
JF - Journal of Histotechnology
IS - 1
ER -