Ab92565

Immunohistochemical analysis was performed on formalin-fixed, deparaffinized tissue sections following heat-induced epitope retrieval^{15 (link)}. The staining was graded as positive when ≥ 10% of tumor cells were reactive, as described in a previous study^{16 (link)}. Appropriate tissues were used as positive and negative controls, respectively. The five candidate markers yielded from microarray analysis were used to validate their expression in clinical samples: BMP8B (N-19, 1:25, sc-6900, Santa Cruz Biotechnology, Dallas, TX, USA), NANOG (polyclonal, 1:25, ab21624, Abcam, Cambridge, MA, USA), HOXA9 (polyclonal, 1:20, ab92565, Abcam), S100A8/MRP8 (7C12/4, 1:25, ab20220, Abcam), and CCR6 (polyclonal, 1:75, ab78429, Abcam).

Immunofluorescent staining was performed as described in a previous study¹⁸ . Briefly, DLBCL (2 × 10^{6 (link)}) cells were cultured in 6-well plates. After cytospinning at 350 rpm for 15 min, cells were transferred onto poly-L-lysine-coated glass slides for immunofluorescence staining. Formaldehyde (100 μl, 4%) was added to fix cells at room temperature for 15 min followed by 0.1% triton for 15 min. After washing with PBS, 3 drops of background-reducing reagent (Dako, S3022, Carpinteria, CA, USA) were added. The cells were incubated with 50–100 μl primary antibodies at 4 °C overnight and then incubated at room temperature in the dark for 1 h with FITC-conjugated anti-rabbit secondary antibody (1:150; Sigma-Aldrich, St. Louis, MO, USA). Nuclear DNA was stained with 4′-6-diamidino-2-phenylindole (DAPI; 1:1000; Invitrogen, Carlsbad, CA, USA) for 15 min at room temperature in the dark. Finally, the cell signal was detected by fluorescence microscopy. The used primary antibodies included NANOG (polyclonal, 1:20, ab21624, Abcam), and HOXA9 (polyclonal, 1:20, ab92565, Abcam).