Publication protocol
IHC analysis were performed on only one section/patient/condition (medium and timepoints)/marker (total of 40 sections per marker) due to scarcity of available tissue. Briefly, sections on Superfrost Plus slides underwent deparaffinization and rehydration. Endogenous peroxidase activity was blocked by incubation for 30 min with 0.3% H2O2. After washing in deionized water for 5 min, the slides were placed in citrate buffer for 75 min at 98°C. Thereafter, they were washed in 0.05 M Tris-buffered saline (TBS) and 0.05% Triton X-100 (Sigma-Aldrich) and, in order to block non-specific binding sites, they wereincubated at room temperature (RT) with 10% normal goat serum (NGS, Invitrogen) and 1% bovine serum albumin (BSA, Invitrogen) for 30 min (for 50 min for active caspase 3). The primary antibody (diluted to 1/500 for MAGEA4, 1/150 for Ki67, 1/200 for anti-Müllerian hormone (AMH), 1/150 for Glial cell line-derived neurotrophic factor (GDNF), 1/100 for the androgen receptor (AR), 1/400 for steroidogenic acute regulatory protein (STAR), 1/200 for active caspase 3, 1/4000 for BOLL, 1/4000 for SYCP3, 1/2000 for SOX9, 1/160 for Angiotensin-converting enzyme (ACE) and 1/2000 for Protamine 2, summarized in Table Table33) was added to the sections and incubated overnight at 4°C in a humidified chamber (60 min for active caspase 3). Regarding double IHC for Ki67-MAGEA4, sections immunostained with anti-Ki67 were washed in HCl 0.1 M for 60 min, followed by washing in distilled water for 5 min and then in 0.05 M TBS and 0.05% Triton X-100 three times for 2 min each. Non-specific antibody binding was blocked by incubation of samples in 10% NGS and 1% BSA for 30 min at RT. MAGEA4 antibody was added to the samples and incubated at 4°C overnight in a humidified chamber.
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