Lymphoma Tissue Specimens
Deidentified human tissue sections of archived formalin-fixed paraffin-embedded (FFPE) blocks were obtained from the Veteran Affairs Medical Center in Decatur, Georgia. Tissue slices (approximately 5 µm thick) were sectioned and placed on positively charged glass slides. The slides were preheated at 60–65 °C for 15 min and then went through the steps of deparaffinization using xylene. Hydration of the slides was performed using a series of ethanol solutions of decreasing concentrations (100%, 95%, 80%, and 70%, twice for each concentration, 2 min in each step). Antigen retrieval was performed using a decloaking chamber (125 °C for 30 s, then 90 °C for 10 s) with common decloaking buffers (Biocare Medical, Concord, CA). The slides were cooled in the decloaking buffer for 20 min, washed in DI water, and stored in 1×PBS plus buffer (containing 0.05% Tween 20).
Multiplexed QD Immunostaining
Multiplexed QD staining was performed, at room temperature, using an automated tissue processing and staining instrument (Nemesis 7200, Biocare Medical, Concord, CA). The use of this robotic system reduced slide-to-slide variations and allowed staining experiments in a high-throughput manner. In the preprogrammed procedure, the slide surface was blocked by 2% BSA/5% goat serum/1×PBS for 30 min at room temperature. The slides were incubated with a mixture of the primary antibodies CD30 (mouse monoclonal, clone Ber-H2, 1:25 dilution, Dako) and Pax5 (rabbit polyclonal, RB-9406-P1, 1:25 dilution, Thermo Fisher Scientific Inc., Fremont, CA) for 1 h. After washing with 1×PBS plus buffer twice, a mixture of the secondary antibody conjugated Qdots (goat antirabbit QD655 and goat antimouse QD605, Invitrogen) was applied to the slides for 2 h. The slides were washed with 1× PBS plus buffer three times. Then, another cycle of QD immunostaining was performed for two additional antigens, using a mixture of the primary antibodies CD15 (mouse monoclonal, clone C3D-1, 1:20 dilution, Dako, Carpinteria, CA) and CD45 (rabbit polyclonal, sc-25590, 1:25 dilution, Santa Cruz Biotechnology Inc., Santa Cruz, CA) and a mixture of goat antirabbit QD565 and goat antimouse QD525 (Invitrogen, Carlsbad, CA). Then, 4′,6-diamidino-2-phenylindole (DAPI) counterstaining (100 ng/mL) was performed, followed by washing with DI water. The slides were dehydrated and cover-slipped using mounting media (Ecomount, Biocare Medical, Concord, CA).
Spectral Imaging and Data Analysis
A multispectral imaging system (Nuance, CRI, Woburn, MA) was mounted on an inverted fluorescence microscope (Olympus I×71, Olympus America Inc., Center Valley, PA) for wavelength-resolved imaging and data acquisition. Near-UV excitation at 350 nm was obtained with a mercury lamp, and a long-pass filter was used to pass the QD fluorescence signals. A series of images (called image stacks or image cubes) were captured in the wavelength range of 500–800 nm at 10 nm increments with a liquid-crystal tunable filter. A spotted array of QDs on the glass slide was used to construct a library of individual QD spectra. Tissue background (autofluorescence) spectra were obtained from control (unstained) specimens.
Consecutive slides from the same tissue block were stained using standard DAB chromagen immunohistochemistry. One protein biomarker was stained on one tissue section. Briefly, after tissue pretreatment as described above and subsequent endogenous enzyme blocking for 15 min, the slides were loaded into the same autostaining machines. The MACH-4 detection system (Biocare Medical, Concord, CA) was used for enhanced sensitivity. The routine counterstaining step with hematoxylin was omitted so that monocolor images from control slides could be used for comparison. Images of IHC-stained tissues were acquired using a color CCD camera attached to an inverted Olympus microscope.