5-methyl cytosine (5mC) IHC stainings

For 5mC immunohistochemical (IHC) stainings, archived embedded testicular tissues from neonatal (n = 3), 4-months-old (n = 3), 8-months-old (n = 3), and adult marmosets (n = 3) were randomly selected and tissue sections were prepared. Tissue sections were deparaffinized in AppliClear (A4632,2500; Appli Chem GmbH) and rehydrated in a descending ethanol series. Antigen retrieval was performed for 10 min at approximately 80°C in citrate buffer. Endogenous peroxidases were inactivated by incubation with hydrogen peroxide (3%). Blocking for unspecific binding was performed with TBS containing 25% chicken serum and 0.5% bovine serum albumin (BSA) for 30 min. Detection of 5mC positive cells was performed with the primary mouse-anti-5mC antibody (C15200081, Clone: 33D3, Lot GF-004, Diagenode, dilution: 1:100), the secondary chicken-anti-mouse IgG-Biotin antibody (sc-2985, Santa Cruz Biotechnology, dilution 1:100), and streptavidin conjugated with HRP (S5512, Sigma-Aldrich, dilution: 1:500). 3,3′-Diaminobenzidine (0.5 mg/ml in TBS + 0.1% hydrogen peroxide) was used to visualize HRP labeled cells. A counterstain with Mayer's hematoxylin solution (1.09249.0500, Merck-Millipore) was performed to allow morphological identification of different testicular cell types. After dehydration in an ethanol series and AppliClear, the stained sections were mounted with Merckoglas (1.03973.0001, Merck). For each staining, negative controls, which were incubated with unspecific mouse IgGs, were performed.