Palm robot microbeam

Frozen lymph node sections positioned on membrane-covered microdissection slides were immunostained for CD30 with the anti-CD30 antibody BerH2 (Dako). Single CD30⁺ B cells were microdissected into 20 µl of 1 x PCR buffer using the PALM Robot MicroBeam laser microdissection system (Zeiss, Oberkochen, Germany) as previously described (11 (link)). Microdissected pieces of membrane not covered by tissue and tubes only carrying PCR buffer served as negative controls.

Ten consecutive 8-μm-thick nuclear fast red-stained representative sections of the tumors were subjected to laser-assisted microdissection on a PALM Robot MicroBeam laser microdissection system (Zeiss, Thornwood, NY, USA), as previously described.^{24 (link)} First, non-neoplastic cells, including inflammatory cells, stroma, and normal breast, were ablated. We subsequently microdissected only histologically unequivocal neoplastic cells from each sample under a microscope. Tissue was microdissected directly into extraction buffer, and DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA) and quantified with the Qubit Fluorometer (Invitrogen, Life Technologies, Norwalk, CT, USA).