DOUBLE-LABELING FOR KERATIN AND CLASS-III BETA-TUBULIN WITHIN CULTURED RETINAL-PIGMENT EPITHELIAL-CELLS - COMPARISON OF CHROMOGENS TO YIELDMAXIMUM RESOLUTION OF 2 STRUCTURAL PROTEINS WITHIN THE SAME CELL

Previous attempts at colocalization of 2 distinct antigens within the same cell have met with various limitations. Either the double-labelin g preparation was not permanent and required a fluorescent microscope (immuno-fluorescence) or 1 chromogen would totally or partially obscur e 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 II I beta-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'-diaminobenzidi ne-HC1 (DAB) or True Blue, HistoMark Blue/DAB, 3-amino-9-ethylcarbazol e (AEC) or HistoMark Orange, and HistoMark Orange/HistoMark Black or T rue Blue. It was unimportant which chromogen was used to localize each protein, but the order in which the chromogens were developed was imp ortant since some chromogens would wash out or diminish in clarity thr ough 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.