4 6 diamidino 2 phenylindole (dapi)

The translocation t(11;14)(q13;q32), cyclin D1 (CCND1)/immunoglobulin heavy chain (IGH), is a hallmark for MCL and was found in most of the cases (32 (link)). Recently, it was reported that some MCL of cyclin D1 negative had translocation of cyclin D2 or cyclin D3 with IGK or IGL partner (34 (link), 35 (link)). Therefore, the fluorescence in situ hybridization (FISH) technique was used to detect the translocation/gene rearrangement of cyclin D1, cyclin D2, and cyclin D3. FFPE slides were deparaffinized with Hemo-De (Thermo Fisher Scientific, Waltham, MA, USA), rehydrated with gradient alcohols, pretreated with 10-fold diluted epitope retrieval solution (Dako North America Inc., Carpentaria, CA, USA) at 97°C for 25 min, digested with pepsin, and then hybridized respectively with dual color and dual fusion probe for t(11;14)(q13;q32) and break-apart probes for cyclin D2 and cyclin D3 (Abbott Molecular, Des Plaines, IL, USA) overnight. The next day, the slides were washed to remove excess FISH probes and counterstained with DAPI (Abbott Molecular, Des Plaines, IL, USA). FISH images were captured and analyzed by BioView Duret FISH analysis system (Billerica, MA, USA).

Cells were collected and then suspended in PBS (Sigma-Aldrich, USA). Carnoy’s Solution (3:1 methanol:acetic acid; Sigma-Aldrich, USA) was added in a 10:1 ratio with the sample suspension, then centrifuged for 5 min at 1500 rpm (300 g). The cell pellet was resuspended in Carnoy’s Solution, incubated for 15 min at –20 °C, and then centrifugation was repeated. Cold 10-μl drops were placed on pre-coated fluoro slides (Thermo Scientific, USA) and warmed to 52 °C. Slides were dehydrated, then decondensed in 0.5 M NaOH (Sigma-Aldrich, USA) solution for 1 min and then dehydration was repeated. Slides were then treated with 4 μl AneuVysion Multicolor DNA Probe (Vysis CEP 18/X/Y; Abbott Molecular, USA), and then incubated in a 37 °C humidified chamber for a minimum of 16 h. Following incubation, slides were rinsed in 0.3% NP 40 in 0.4× SSC warmed to 73 °C for 2 min followed by 0.1% NP 40 in 2× SSC for 10 s. Once dry, slides were counterstained with DAPI (Abbott Molecular, USA) and coverslipped. Slides were analyzed for diploidy by examining chromosomes X, Y, and 18 using DAPI, FITC, TxRed, and Aqua channels on an Olympus BX 61 fluorescence microscope and imaged using HCImage software (Hamamatsu Photonics, Japan). Human sperm was used as a positive control for analysis. Slides were stored between analyses and for the long-term at –20 °C.