The FA triple staining immunofluorescence method has been previously described [22 (link)]. Briefly, FFPE tumor tissue was cut at 4 microns, placed on positively charged slides, and stained with hematoxylin and eosin. Additional sections for immunofluorescence staining were placed in a 60 °C oven for one hour, cooled, deparaffinized, and rehydrated through xylenes and graded ethanol solutions to water in standard fashion. Antigen retrieval was performed by placing slides in Dako’s TRS antigen retrieval solution (Dako) in a calibrated vegetable steamer (Black and Decker) at 94 °C for 25 min. Slides then were placed on a Dako Autostainer for automated staining. The tissue sections were incubated with a primary antibody cocktail of rabbit polyclonal FANC-D2 antibody at a dilution of 1:1,000 and a monoclonal anti-Ki67 mouse antibody (Dako) at a dilution of 1:150, for one hour at room temperature. Sections then were incubated with a secondary antibody (FITC conjugated to anti-rabbit IgG and Alexa fluor 594 donkey anti-mouse IgG, Invitrogen) at 1:1,000 for one hour at room temperature. The sections were mounted on glass slides using a 4′ 6-diamidino-2-phenylindole (DAPI)-containing embedding medium (Vysis Dapi 1, Abbott Laboratories). The slides were analyzed under a Nikon E-400 fluorescence microscope [22 (link)].
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