X0943

Formalin-fixed, paraffin-embedded (FFPE) sections from primary MCC tumors were prepared and analyzed as previously described (55 (link)). The primary antibodies were cytokeratin 20 (CK20) antibody (dilution 1:50 [Dako]), MCPyV LT antibody CM2B4 (dilution 1:125 [Santa Cruz Biotechnology]), and anti-stathmin 1 antibody (dilution 1:250 [Abcam]). An isotype-matched irrelevant antibody was used as a negative control on serial sections of tissues in parallel, Dako X0943 was used for the CK20 primary antibody, and the rabbit polyclonal isotype control antibody (Abcam) was used to match the stathmin primary antibody. Sections were incubated with appropriate secondary antibodies labeled with different fluorochromes: Alexa Fluor 488 IgG2B and Alexa Fluor 633 IgG2A [Invitrogen] and IgG (H+L)-tetramethyl rhodamine isocyanate (TRITC) (Jackson ImmunoResearch). Nuclear counterstaining was with bis-benzimide (Invitrogen). All slides were mounted with Immu-Mount, and images were captured with a Zeiss LSM 510 confocal microscope (56 (link)).

For immunohistochemical analysis, micromasses were fixated for 2 h in 2 % paraformaldehyde in phosphate-buffered saline (PBS)/1 mM CaCl_2, dehydrated and embedded in paraffin. Sections 7-μm thick were deparaffinized, rehydrated and incubated with antibodies 11-fibrau (1:100 for 60 minutes) (clone D7-fib, Imgen, distributed by ITK Diagnostics, Uithoorn, The Netherlands), mouse anti-human cluster of differentiation 68 (CD68) (1:100 for 60 minutes) (M0814, DAKO, Heverlee, Belgium) or control mouse IgG2ak (X0943, DAKO) and IgG1k (X0931, DAKO) respectively. Endogenous peroxidase activity was blocked with 3 % H₂O₂ (Merck Millipore, Amsterdam, The Netherlands) in methanol. Subsequently, the sections were incubated with the secondary horseradish peroxidase (HRP)-conjugated rabbit-anti-mouse IgA/G/M (1:200 for 60 minutes) (P0260, DAKO). Peroxidase was developed with diaminobenzidine and counterstained with hematoxylin for 60 seconds.

The absence of BM infiltration with neoplastic cells was confirmed by an immunocytochemistry system (cat. K0673, Dako, Carpinteria, CA, USA) and cell morphology analysis was performed by the Pappenheim technique. Aspirates from BM were stained with antibodies (Abs) against epithelial membrane antigen (cat. M0613, Dako, Carpinteria, CA, USA) and cytokeratins AE1–AE3 (cat. IR053, Dako, Carpinteria, CA, USA). Samples were considered positive for metastasis only if cells expressed EMA and cytokeratins AE1–AE3 and if cells were morphologically malignant. Isotype controls (cat. X0943, Dako, Carpinteria, CA, USA, and cat. 08-6599, ZYMED Laboratories, South San Francisco, CA, USA) were run in parallel using the same concentration of each Ab tested [37 (link)]. Experiments were repeated two times for each sample.