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88 C H A P T E R 6 The buffer was first heated until boiling in the microwave for 3 min at maximum Watt. After cooling down for 2 min at room temperature, the buffer was heated again in the microwave for 12 min at minimum Watt. Non-specific binding sites were blocked with 5% BSA/PBS/0.5% Triton X-100. This was followed by overnight incubation at 4 °C with Anti- phospho-Histone H2A.X diluted 1:150 in 1% BSA/PBS/0.05% Tween.After incubation of the primary antibody, the same steps were performed as for the detection of spermatogonia with MAGE A3/A4. For the detection of round spermatids and spermatozoa, slides were stained for the presence of their Acrosin cap using rabbit polyclonal Acrosin antibody (ThermoFisher, PA5-61804). Antigen retrieval was carried out by boiling tissue sections in 0.01 M sodium citrate buffer (tri-sodium citrate dihydrate Na 3 C 6 H 5 O 7 .2H 2 O, pH 6.0). The buffer was first heated until boiling in the microwave for 3 min at maximum Watt. After cooling down for 2 min at room temperature, the buffer was heated again in the microwave for 10 min at minimum Watt. Subsequently, the buffer was cooled down for 10 min at room temperature and placed under running tap water. After these steps, a standard immunohistochemical preparation protocol was followed, as described above. For all three antibodies, slides with testicular tissue from a prostate cancer patient with normal spermatogenesis served as a positive control. Negative controls were carried out by replacing the first antibody by isotype IgG (Supplementary Figure 1). Immunohistochemical analysis The immunohistochemically stained slides were examined using a bright field microscope (Olympus BX41) and assessed on the presence of the specifically targeted germ cell type. Outcome was then used to validate the Modified Johnsen’s scoring of the histologically analyzed slide of that same specimen. Results from the immunohistochemically stained slides were preferred if there was a difference between the two. Statistical analyses After completion of histological and immunohistochemical analyses, results were linked to clinical data and descriptive analyses were conducted for the total cohort and the six subgroups. Data are presented as means (SD) when normally distributed, as medians with interquartile ranges (IQR) when non-normally distributed, or as numbers with percentage. Progress of spermatogenesis, determined by the presence of the most advanced germ cell type per orchiectomy specimen, was used as main outcome measurement (no germ cells, spermatogonia, spermatocytes, round spermatids, or spermatozoa). Secondary outcome measurements included mean Johnsen score per orchiectomy specimen, the degree of hyalinization, and presence of a lumen. To assess the possibilities for fertility preservation three categories were defined: preservation of spermatozoa; preservation of spermatogonial stem cells for those with