P-EMBL-111 - P-EMBL-111

Testicular tissue samples were obtained directly after orchidectomy and macroscopic pathological evaluation. The use of the orchidectomy samples was approved by the Regional Committee for Medical Research Ethics in Denmark. Samples of homogeneous overt tumors (seminoma or EC) and of testicular parenchyma preserved in the vicinity of a tumor were excised. Each testicular sample was divided into several tissue fragments: 2 - 3 fragments were snap-frozen at -80C for nucleic acid extraction, several adjacent fragments were fixed overnight at 4C in Stieve's fluid or paraformaldehyde (PFA), and embedded in paraffin. Fixed sections were subsequently stained with haematoxylin and eosin (HE) or with an antibody against placental-like alkaline phosphatase (ALPP or PLAP) for histological evaluation. Most samples of testicular parenchyma contained variable numbers of tubules with CIS cells. Samples where no tubules with CIS were found and complete spermatogenesis was present were used as a normal reference. In an attempt to limit the uncertainty about cellular differences between fragments from the same patient, we also made a direct print of the frozen fragment that specifically was used for RNA isolation. After staining with ALPP (PLAP) antibody, we could roughly confirm the approximate proportion of tubules with CIS as established by histology in adjacent tissue fragments. One sample from tissues containing approximately 50%, 75%, and 100% tubules with CIS cells, respectively, together with fragments with no neoplastic changes and preserved normal spermatogenesis (serving as the common reference) were chosen for microarray analysis.
Experiment E-EMBL-2