Hemo de

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).

Corneas were paraffin-embedded and cut in 5μm serial sections [28 (link)], then the paraffin was removed by a xylene substitute (Hemo-De; Thermo-Fisher Scientific, Darmstadt, Germany). The immunohistochemistry procedure was performed as previously described [24 (link)] by BenchMark Automated IHC/ISH slide staining system (BenchMarkVentana, Tucson, AZ, USA). Sections were incubated with specific anti-ki67 (Rabbit polyclonal anti-Ki67 antibody; concentration 5 μg/mL; ab155807 Abcam) and anti-VEGF (Mouse monoclonal anti-VEGF antibody; 1:100; sc-57496, Santa Cruz) antibodies. After washes with PBE, the section was incubated with biotin-conjugated secondary antibodies and avidin-biotin-peroxidase complex (DBA, Milan, Italy). Specific antigens in each section were located with 3,3′diaminoenzidine (DAB) reaction, then slides were counterstained with hematoxylin. An expert pathologist analyzed the immunostaining (intraobserver variability 5%). Ki67 and VEGF antigenic expression was measured and calculated by Leica IM500 and statistics program Leica QWIN (Leica, Wetzlar, Germany). Four distinct preparations for each corneal sample were done and 18 fields of view were analyzed in each preparation for a total area of 1.35631e ± 0.003 μm².

A section of each tumor was fixed in 10% formalin for IHC determinations. Sections of 3–4 μm were cut from each sample by deparaffinizing and dehydrating using Hemo-De (Fisher Scientific, Pittsburgh, PA, USA), followed by an alcohol series and washing in phosphate-buffered saline (PBS), pH 7.0. The slides were then incubated in 3% H₂O₂ for 20 min at room temperature to block peroxidase, and antigens were retrieved by boiling the slides in a microwave oven in 50 mM citrate buffer, pH 6.4. After blocking with goat serum, the slides were incubated with PCNA, Ki67, and cleaved caspase-3 antibodies (Google Biological Science and Technology Co., Ltd., Wuhan, China). The slides were counterstained with hematoxylin. H&E stained sections were also made. Apoptosis was detected by the TUNEL assay using the DeadEnd^TM Colorimetric TUNEL System (Promega, Madison, WI, USA) per manufacturer’s protocol. Three random microscopic fields were captured for each tumor section at 200× magnification.