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87 HISTOLOGICAL STUDY ON THE INFLUENCE OF PUBERTY SUPPRESSION AND HORMONAL TREATMENT ON DEVELOPING GERM CELLS 6 Histological analysis Histological examination was conducted using a bright field microscope (Olympus BX41, OM Digital Solutions Americas, Bethlehem, PA, USA). From each specimen, at least 50 seminiferous tubules per slide were analyzed to assess spermatogenesis by determining the most advanced germ cell type from each seminiferous tubular cross section based on their location within the tubule and nuclear morphology. The Modified Johnsen’s scoring system was used to assign a score to each tubule, and per slide a mean Johnsen’s score was calculated. The Modified Johnsen’s scoring system involves a 10-point Likert scale where score 1 corresponds to complete sclerosis without recognizable seminiferous epithelium, and score 10 implies the presence of more than 10 elongated spermatids without immature and apoptotic cells in the lumen (Supplementary Table 1). 112 After assessment of spermatogenesis, overall testicular histology was assessed including the presence of a lumen in the seminiferous tubules and rate of seminiferous tubule hyalinization. The lumen was categorized as open, half-open or absent. Hyalinization was defined as a hyaline area separating the peritubular layer from the basal membrane of the seminiferous tubule. Preparation for immunohistochemistry In order to validate our findings, a second slide of each specimen was analyzed using immunohistochemistry. The primary antibodies were chosen based on the most advanced germ cell type that was identified during histological analysis, or on uncertainty regarding the presence of a germ cell type. For the detection of spermatogonia, slides were stained for spermatogonial marker MAGE-A3/A4 using mouse monoclonal Anti-Mage A3/A4 antibody (clone 57B; Merck Millipore, Germany). Endogenous peroxidase activity was inactivated with 0.3% H₂O₂/ PBS for 10 min at room temperature in the dark. Non-specific binding sites were then blocked with Superblock (ScyTek Lab, USA) for 1 hour at room temperature in a humid slide box. Sections were subsequently incubated overnight at 4 °C with Anti-Mage A3/ A4 antibody diluted 1:2000 in BrightDiluent (Immunologic, the Netherlands). The next day, all slides were washed three times with phosphate buffered saline (PBS) followed by 30 min incubation with Powervision goat-anti Mouse/Rabbit poly-horseradish peroxidase (DPVO110HRP, Immunologic, the Netherlands) secondary antibody at room temperature. After washing, the signal was visualized using Bright-DAB (3,3’-diaminobenzidine, Immunologic, the Netherlands) after which the sections were counterstained with Mayer’s haematoxylin. Finally, after dehydration in increasing ethanol concentrations and xylene, the slides were encapsulated with glass coverslips using Entellan® (Merck Millipore, Germany) for further microscopic analysis. For the detection of spermatocytes, slides were stained for γ H2AX using mouse monoclonal Anti-phospho-Histone H2A.X (Merck Millipore, Germany) antibody. Antigen retrieval was carried out by boiling tissue sections in Tris-EDTA buffer (10mM Tris, 1mM EDTA, pH=9.0).